技术领域Technical field
本发明涉及生物技术领域，涉及基因诊断领域，涉及一种分级模型及其应用，涉及一种用于检测淋巴瘤、淋巴转移癌良恶性程度的分级模型及其应用，具体涉及一组印记基因在检测淋巴瘤、淋巴转移癌良恶性程度的分级模型及其组成的装置。The present invention relates to the field of biotechnology, to the field of genetic diagnosis, to a grading model and its application, to a grading model for detecting the benign and malignant degree of lymphoma and lymphatic metastasis cancer and its application, and specifically to a group of imprinted genes in A grading model for detecting the benign and malignant degree of lymphoma and lymphatic metastasis cancer and a device thereof.
背景技术Background technique
淋巴瘤是起源于淋巴造血系统的恶性肿瘤，主要由病毒感染、免疫缺陷、电离辐射和化学药物等原因引起，好发于20-40岁的青壮年。2012年恶性淋巴瘤全球新增病例38.6万例，死亡20.0万例，中国新诊断病例约8.8万例，死亡5.2万例，并且近年来仍然在以每年4％的速度增长。淋巴瘤可以发生在人体的任何部位，早期症状仅为无痛性淋巴结肿大，容易被病人忽略，也容易与感染引起的反应性淋巴结增生混淆。由于淋巴瘤是一大类异质性很高的肿瘤的集合，按照WHO的病理分类标准，可以分为上百种类型，各种分型之间在病理表现和预后都具有很大的差异，因此淋巴瘤的诊断和治疗都比较困难。淋巴瘤总体上可以分为高度侵袭性、中等侵袭性和惰性淋巴瘤，高度侵袭性的淋巴瘤5年生存率通常不到30％，而某些类型的惰性淋巴瘤5年生存率可以达到90％，一些中等侵袭性的淋巴瘤如果能够早期发现和治疗，生存期可以达到5-10年甚至更长，因此开发一种准确率更高的恶性淋巴瘤早期检测技术具有重大意义。Lymphoma is a malignant tumor originating from the lymphatic hematopoietic system. It is mainly caused by viral infection, immune deficiency, ionizing radiation and chemical drugs. It mostly occurs in young adults aged 20-40 years old. In 2012, there were 386,000 new cases of malignant lymphoma worldwide and 200,000 deaths. In China, there were approximately 88,000 newly diagnosed cases and 52,000 deaths. In recent years, the number is still growing at an annual rate of 4%. Lymphoma can occur in any part of the human body. The early symptoms are only painless lymph node enlargement, which is easily ignored by patients and can also be easily confused with reactive lymph node hyperplasia caused by infection. Since lymphoma is a collection of highly heterogeneous tumors, it can be divided into hundreds of types according to the WHO pathological classification standards. There are great differences in pathological manifestations and prognosis among various types. Therefore, the diagnosis and treatment of lymphoma are difficult. Lymphoma can generally be divided into highly aggressive, moderately aggressive and indolent lymphoma. The 5-year survival rate of highly aggressive lymphoma is usually less than 30%, while the 5-year survival rate of some types of indolent lymphoma can reach 90%. %. If some moderately aggressive lymphomas can be detected and treated early, the survival period can reach 5-10 years or even longer. Therefore, it is of great significance to develop a more accurate early detection technology for malignant lymphoma.
除淋巴瘤外，淋巴结肿大也可能由癌症转移引起，肺癌、乳腺癌、食道癌、甲状腺癌、结直肠癌和膀胱癌等多种癌症都会发生淋巴转移。目前病理上判断淋巴结中是否有转移的癌细胞通常是依据形态观察，但是在癌转移的早期，淋巴结中癌细胞的数量还较少，通过形态观察可能会遗漏，因此开发一种准确鉴别淋巴结中是否有癌细胞转移的技术，可以帮助医生确定手术切除范围和淋巴结清扫范围，并知道后续治疗方案的选择，对降低手术后的复发率、延长生存期具有重大意义。In addition to lymphoma, lymph node enlargement may also be caused by cancer metastasis. Lymphatic metastasis occurs in various cancers such as lung cancer, breast cancer, esophageal cancer, thyroid cancer, colorectal cancer, and bladder cancer. At present, pathological judgment of whether there are metastatic cancer cells in lymph nodes is usually based on morphological observation. However, in the early stage of cancer metastasis, the number of cancer cells in lymph nodes is still small and may be missed through morphological observation. Therefore, a method to accurately identify lymph nodes is developed. Whether there is cancer cell metastasis technology can help doctors determine the scope of surgical resection and lymph node dissection, and know the choice of subsequent treatment options, which is of great significance to reducing the recurrence rate after surgery and prolonging survival.
传统病理学对细胞的良恶性诊断是基于细胞的大小，形态，浸润性和周边细胞组织的关系来作出判断的。它对细胞(癌症)的早期变化的发现有很大的局限性，因此细胞分子水平的癌症诊断方法，一度成为研究热点。随着人们在分子生物学领域的不断深入研究，越来越多的分子检测技术被运用到癌症诊断中。Traditional pathology diagnosis of benign and malignant cells is based on the size, shape, infiltration and relationship between cells and surrounding cells and tissues. It has great limitations in discovering early changes in cells (cancer). Therefore, cancer diagnosis methods at the cellular and molecular levels have become a research hotspot. As people continue to conduct in-depth research in the field of molecular biology, more and more molecular detection technologies are being used in cancer diagnosis.
癌症的产生是随时间推移而累积的表观遗传改变和基因上的变异所导致的不受控制的细胞生长/分裂。传统病理学诊断根据细胞和组织的大小，形态和结构上的变异，从而做出淋巴瘤良恶性判断。随着分子生物学的发展与深入，越来越多的分子检测技术被应用于恶性淋巴瘤症的检测。从癌症的发展过程分析，分子层面的改变(表观遗传学和基因学)远早于细胞形态和组织结构的变异。所以分子生物学检测对癌症早期的检测更敏感。Cancer arises from uncontrolled cell growth/division caused by epigenetic changes and genetic mutations that accumulate over time. Traditional pathological diagnosis is based on the size, morphology and structural variation of cells and tissues to judge whether lymphoma is benign or malignant. With the development and deepening of molecular biology, more and more molecular detection technologies are being used in the detection of malignant lymphoma. From the analysis of the development process of cancer, changes at the molecular level (epigenetics and genetics) occur much earlier than changes in cell morphology and tissue structure. Therefore, molecular biology testing is more sensitive for early detection of cancer.
基因组印记是表观遗传学中基因调控的一种方式。其特点是，通过甲基化来自特定亲代的等位基因，使某个基因只有一个等位基因表达，而另一个则陷入基因沉默状态。该种类的基因，被称为印迹(记)基因。印迹缺失是印迹基因去甲基化导致沉默状态的等位基因被激活并且开始基因表达的一种表观遗传改变。大量研究表明，该现象(印迹缺失)普遍存在于各类癌症并且发生时间早于细胞和组织形态改变。与此同时，在健康细胞中，印迹缺失比例极低，与癌细胞成鲜明对比。所以，印迹基因的甲基化状态可以作为病理标记，通过特定分子检测技术，对细胞异常状态进行分析。Genomic imprinting is a form of gene regulation in epigenetics. Its characteristic is that by methylating alleles from a specific parent, only one allele of a certain gene is expressed, while the other is silenced. This type of gene is called an imprinted gene. Imprinting loss is an epigenetic change in which demethylation of an imprinted gene causes the silent allele to become activated and initiate gene expression. A large number of studies have shown that this phenomenon (loss of imprinting) is prevalent in various types of cancer and occurs earlier than changes in cell and tissue morphology. At the same time, in healthy cells, the proportion of imprinting loss was extremely low, in sharp contrast to cancer cells. Therefore, the methylation status of imprinted genes can be used as a pathological marker to analyze abnormal cell status through specific molecular detection technologies.
基于上述原因，目前的恶性淋巴瘤诊断需要新的检测系统和检测模型，基于患者活检样本，解析恶性淋巴瘤在细胞层面上存在的分子标记物变化，以此提供更精确的预诊和诊断信息。For the above reasons, the current diagnosis of malignant lymphoma requires new detection systems and detection models. Based on patient biopsy samples, the changes in molecular markers present in malignant lymphoma at the cellular level are analyzed to provide more accurate prediagnosis and diagnostic information. .
发明内容Contents of the invention
针对现有技术的不足及实际的需求，本发明提供了一种用于检测淋巴瘤、淋巴转移癌良恶性程度的分级模型及其应用，该检测装置和模型是用于单细胞和组织水平下早期直观地观察淋巴瘤和淋巴转移癌的印记(迹)基因的变化从而判断淋巴瘤的良恶性程度以及癌症是否发生淋巴转移。In view of the shortcomings of the existing technology and actual needs, the present invention provides a grading model for detecting the benign and malignant degree of lymphoma and lymphatic metastasis cancer and its application. The detection device and model are used at the single cell and tissue levels. Early and intuitive observation of changes in imprinted (trace) genes of lymphoma and lymphatic metastasis cancer can help determine the benign and malignant degree of lymphoma and whether the cancer has lymphatic metastasis.
为达到上述目的，本发明采用以下技术方案：In order to achieve the above objects, the present invention adopts the following technical solutions:
第一方面，本发明提供了一种用于淋巴瘤的印记基因分级模型，所述模型通过计算印记基因的总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在淋巴瘤中的变化对印记基因的表达状态进行分级；In a first aspect, the present invention provides an imprinted gene grading model for lymphoma. The model calculates the total expression of imprinted genes, the expression of imprinted gene deletion and the abnormal expression of imprinted gene copy number in lymphoma. Changes grade the expression status of imprinted genes;
其中，所述印记基因为Z1、Z8、Z11或Z16中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z8为Dcn，所述印记基因Z11为Grb10，所述印记基因Z16为Snrpn/Snurf。Wherein, the imprinted gene is any one or a combination of at least two of Z1, Z8, Z11 or Z16, the imprinted gene Z1 is Gnas, the imprinted gene Z8 is Dcn, the imprinted gene Z11 is Grb10, so The imprinted gene Z16 is Snrpn/Snurf.
本发明中，发明人发现通过计算Z1、Z8、Z11和Z16中任意一个印记基因在淋巴瘤中的印记基因缺失表达量和印记基因拷贝数异常表达量，能够实现对恶性淋巴瘤和淋巴转移癌的诊断。In the present invention, the inventor found that by calculating the expression amount of imprinted gene deletion and the abnormal expression amount of imprinted gene copy number in lymphoma of any imprinted gene Z1, Z8, Z11 and Z16, it is possible to realize the diagnosis of malignant lymphoma and lymphoid metastasis cancer. diagnosis.
根据本发明，针对淋巴瘤的印记基因分级模型，若初步检测只检测一个印记基因，可以检测Z1、Z8或Z16中的任意一个，优选为Z1。According to the present invention, for the imprinted gene hierarchical model of lymphoma, if only one imprinted gene is detected in the preliminary test, any one of Z1, Z8 or Z16 can be detected, preferably Z1.
本发明中，发明人发现，通过计算Z1、Z8或Z16中任意一个印记基因在淋巴瘤中的印记基因缺失表达量和印记基因拷贝数异常表达量，诊断敏感度可以达到57.4％以上；若单独检测一个Z1印记基因，对恶性淋巴瘤的诊断敏感度可以达到66.1％，若单独检测一个Z8印记基因，对恶性淋巴瘤的诊断敏感度可以达到57.4％，若单独检测一个Z16印记基因，对恶性淋巴瘤的诊断敏感度可以达到62.0％。In the present invention, the inventor found that by calculating the expression amount of imprinted gene deletion and the abnormal expression amount of imprinted gene copy number in lymphoma of any imprinted gene Z1, Z8 or Z16, the diagnostic sensitivity can reach more than 57.4%; if alone By detecting a Z1 imprinted gene, the diagnostic sensitivity for malignant lymphoma can reach 66.1%. If a Z8 imprinted gene is detected alone, the diagnostic sensitivity for malignant lymphoma can reach 57.4%. If a Z16 imprinted gene is detected alone, the diagnostic sensitivity for malignant lymphoma can reach 57.4%. The diagnostic sensitivity of lymphoma can reach 62.0%.
根据本发明，针对淋巴瘤的印记基因分级模型，若检测印记基因的两个印记基因的组合，所述模型计算印记基因的方法为：计算Z1、Z8或Z16中的任意两个，优选为Z1和Z8的组合，Z8和Z16的组合。According to the present invention, for the imprinted gene grading model of lymphoma, if a combination of two imprinted genes of an imprinted gene is detected, the method of calculating the imprinted gene by the model is: calculating any two of Z1, Z8 or Z16, preferably Z1 The combination with Z8, the combination of Z8 and Z16.
本发明中，发明人发现通过计算两个或两个以上的印记基因的总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量可以进一步提高敏感度，检测印记基因Z1、Z8和Z16中的任意两个印记基因的组合，对恶性淋巴瘤的诊断敏感度可以达到76.0％以上，检测Z1和Z8的组合时，对恶性淋巴瘤的诊断敏感度可以达到85.2％，检测Z8和Z16的组合时，对恶性淋巴瘤的诊断敏感度可以达到84.4％以上。In the present invention, the inventor found that the sensitivity can be further improved to detect imprinted genes Z1, Z8 and Z16 by calculating the total expression amount of two or more imprinted genes, the expression amount of imprinted gene deletion and the abnormal expression amount of imprinted gene copy number. The combination of any two imprinted genes in the gene can have a diagnostic sensitivity of more than 76.0% for malignant lymphoma. When detecting the combination of Z1 and Z8, the diagnostic sensitivity for malignant lymphoma can reach 85.2%. When detecting Z8 and Z16, When combined, the diagnostic sensitivity for malignant lymphoma can reach over 84.4%.
根据本发明，针对淋巴瘤的印记基因分级模型，所述印记基因还包括Z4、Z5、Z6、Z11或Z13中的任意一个或至少两个的组合；其中，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z11为Grb10，所述印记基因Z13为Sgce。According to the present invention, for the imprinted gene hierarchical model of lymphoma, the imprinted gene also includes any one or a combination of at least two of Z4, Z5, Z6, Z11 or Z13; wherein, the imprinted gene Z4 is Igf2r, so The imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z11 is Grb10, and the imprinted gene Z13 is Sgce.
根据本发明，针对淋巴瘤的印记基因分级模型，所述模型计算印记基因的方法为：计算印记基因的组合，计算Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的八个印记基因的组合。According to the present invention, for the imprinted gene hierarchical model of lymphoma, the method of calculating imprinted genes in the model is: calculating the combination of imprinted genes, and calculating the eight imprinted genes of Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 The combination.
本发明中，发明人发现在使用Z1、Z8和Z16基因检测的基础上再增加Z4、Z5、Z6、Z11、Z13基因进行联合诊断，不仅有助于增加检测的准确度，而且增加其他探针辅助诊断可以进一步避免假阳性的出现，能够将检测准确度进一步提高，从而能够实现所有淋巴瘤样本的精确分级和判断。In the present invention, the inventor found that on the basis of using Z1, Z8 and Z16 gene detection and adding Z4, Z5, Z6, Z11, Z13 genes for joint diagnosis, it not only helps to increase the accuracy of detection, but also adds other probes Auxiliary diagnosis can further avoid the occurrence of false positives and further improve detection accuracy, thereby enabling accurate grading and judgment of all lymphoma samples.
第二方面，本发明提供了一种用于淋巴转移癌的印记基因分级模型，所述模型通过计算印记基因拷贝数异常表达量在淋巴转移癌中的变化对印记基因的表达状态进行分级；In a second aspect, the present invention provides an imprinted gene grading model for lymphatic metastasis cancer, which classifies the expression status of imprinted genes by calculating changes in the abnormal expression amount of imprinted gene copy numbers in lymphoid metastasis cancer;
其中，所述印记基因为Z1、Z8、Z11或Z16中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z8为Dcn，所述印记基因Z11为Grb10，所述印记基因Z16为Snrpn/Snurf。Wherein, the imprinted gene is any one or a combination of at least two of Z1, Z8, Z11 or Z16, the imprinted gene Z1 is Gnas, the imprinted gene Z8 is Dcn, the imprinted gene Z11 is Grb10, so The imprinted gene Z16 is Snrpn/Snurf.
本发明中，淋巴转移癌包括甲状腺癌、乳腺癌、肺癌、食道癌、胃癌、结直肠癌、胰腺癌、肝癌、膀胱癌、前列腺癌、皮肤癌转移到淋巴系统中。In the present invention, lymphatic metastasis cancer includes thyroid cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, bladder cancer, prostate cancer, and skin cancer that metastasize to the lymphatic system.
本发明中，发明人发现通过计算Z1、Z8、Z11和Z16中任意一个印记基因在淋巴转移癌中的印记基因拷贝数异常表达量，能够实现对恶性淋巴瘤和淋巴转移癌的诊断。In the present invention, the inventor found that by calculating the abnormal expression of imprinted gene copy number of any one of Z1, Z8, Z11 and Z16 imprinted genes in lymphatic metastasis cancer, diagnosis of malignant lymphoma and lymphatic metastasis cancer can be achieved.
根据本发明，针对淋巴转移癌的印记基因分级模型，若初步检测只检测一个印记基因，可以检测Z1、Z11或Z16中的任意一个，优选为Z16。According to the present invention, for the imprinted gene hierarchical model of lymphatic metastasis cancer, if the preliminary detection only detects one imprinted gene, any one of Z1, Z11 or Z16 can be detected, preferably Z16.
本发明中，发明人发现，通过计算Z1、Z11或Z16中任意一个印记基因在淋巴转移癌中印记基因拷贝数异常表达量，诊断敏感度可以达到86.4％以上；若单独检测一个Z1印记基因，对癌症淋巴转移的诊断敏感度可以达到87.1％，若单独检测一个Z11印记基因，对癌症淋巴转移的诊断敏感度可以达到86.4％，若单独检测一个Z16印记基因，对癌症淋巴转移的诊断敏感度可以达到91.3％。In the present invention, the inventor found that by calculating the abnormal expression of imprinted gene copy number in lymphatic metastasis cancer of any imprinted gene Z1, Z11 or Z16, the diagnostic sensitivity can reach more than 86.4%; if one Z1 imprinted gene is detected alone, The diagnostic sensitivity for cancer lymphatic metastasis can reach 87.1%. If a Z11 imprinted gene is detected alone, the diagnostic sensitivity for cancer lymphatic metastasis can reach 86.4%. If a Z16 imprinted gene is detected alone, the diagnostic sensitivity for cancer lymphatic metastasis can reach 86.4%. It can reach 91.3%.
根据本发明，针对淋巴转移癌的印记基因分级模型，若检测印记基因的两个印记基因的组合，所述模型计算印记基因的方法为：计算Z1、Z11或Z16中的任意两个，优选为Z1和Z16的组合，Z11和Z16的组合。According to the present invention, for the imprinted gene hierarchical model of lymphatic metastasis cancer, if a combination of two imprinted genes of an imprinted gene is detected, the method of calculating the imprinted gene by the model is: calculating any two of Z1, Z11 or Z16, preferably The combination of Z1 and Z16, the combination of Z11 and Z16.
本发明中，发明人发现通过计算两个或两个以上的印记基因拷贝数异常表达量可以进一步提高敏感度，检测印记基因Z1、Z11和Z16中的任意两个印记基因的组合，对淋巴转移癌的诊断敏感度可以达到95.5％以上，检测Z1和Z6的组合，Z11和Z16的组合时，对淋巴转移癌症的诊断敏感度可以达到99.0％以上。。In the present invention, the inventor found that the sensitivity can be further improved by calculating the abnormal expression levels of two or more imprinted gene copy numbers, detecting the combination of any two imprinted genes Z1, Z11 and Z16, and detecting lymphatic metastasis. The diagnostic sensitivity of cancer can reach more than 95.5%. When detecting the combination of Z1 and Z6, and the combination of Z11 and Z16, the diagnostic sensitivity of lymphatic metastasis cancer can reach more than 99.0%. .
根据本发明，针对淋巴转移癌的印记基因分级模型，所述印记基因还包括Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z12、Z13、Z14或Z15中的任意一个或至少两个的组合；其中，所述印记基因Z2为Igf2，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z9为Dlk1，所述印记基因Z10为Gatm，所述印记基因Z12为Peg3，所述印记基因Z13为Sgce，所述印记基因Z14为Slc38a4，所述印记基因Z15为Diras3。According to the present invention, for the imprinted gene hierarchical model of lymphatic metastasis cancer, the imprinted genes also include any one or at least two of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14 or Z15 A combination of; wherein, the imprinted gene Z2 is Igf2, the imprinted gene Z3 is Peg10, the imprinted gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, the imprinted gene Z9 is Dlk1, the imprinted gene Z10 is Gatm, the imprinted gene Z12 is Peg3, the imprinted gene Z13 is Sgce, the imprinted gene Z14 is Slc38a4, the imprinted gene Z15 is Diras3.
根据本发明，针对淋巴转移癌的印记基因分级模型，所述模型计算印记基因的方法为：计算印记基因的组合，计算印记基因的组合，计算Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16的十五个印记基因的组合。According to the present invention, for the imprinted gene grading model of lymphatic metastasis cancer, the method of calculating imprinted genes in the model is: calculating the combination of imprinted genes, calculating the combination of imprinted genes, and calculating Z1, Z2, Z3, Z4, Z5, Z6, Z8 , a combination of fifteen imprinted genes of Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
本发明中，发明人发现在使用Z1、Z11和Z16基因检测的基础上再增加Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z12、Z13、Z14、Z15基因进行联合诊断，不仅有助于增加检测的准确度，而且增加其他探针辅助诊断可以进一步避免假阳性的出现，能够将检测准确度进一步提高，从而能够实现所有淋巴转移癌样本的精确分级和判断。In the present invention, the inventor found that on the basis of using Z1, Z11 and Z16 gene detection and adding Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14 and Z15 genes for joint diagnosis, not only It helps to increase the accuracy of detection, and adding other probes to assist diagnosis can further avoid the occurrence of false positives and further improve the accuracy of detection, thereby enabling accurate grading and judgment of all lymphatic metastasis cancer samples.
本发明中，所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，所述印记基因拷贝数异常为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，所述拷贝数异常是由于癌细胞异常地进行基因复制，导致这个基因表达时呈现为三倍体甚至更高的多倍体的情况。In the present invention, the imprinted gene deletion means that after hematoxylin staining of the cells, there are two red/brown markers in the cell nucleus, and the imprinted gene copy number abnormality means that after the cells are hematoxylin stained, there are more than two red/brown marks in the cell nucleus. Brown mark, the copy number abnormality is due to abnormal gene replication in cancer cells, resulting in the expression of this gene as triploidy or even higher polyploidy.
本发明中，所述印记基因与印迹基因同时一个概念，表示同一个意思，可以进行替换。In the present invention, the imprinted gene and the imprinted gene are the same concept, represent the same meaning, and can be replaced.
根据本发明，所述计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：According to the present invention, the formula for calculating the total expression amount of imprinted genes, the expression amount of missing imprinted genes and the abnormal expression amount of imprinted gene copy number is as follows:
总表达量＝(b+c+d)/(a+b+c+d)×100％；Total expression amount=(b+c+d)/(a+b+c+d)×100%;
正常印记基因表达量＝b/(b+c+d)×100％；Normal imprinted gene expression = b/(b+c+d)×100%;
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；Imprinted gene deletion gene expression level (LOI) = c/(b+c+d)×100%;
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；Gene expression amount with abnormal imprinted gene copy number (CNV) = d/(b+c+d)×100%;
其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。Wherein, the a is a cell nucleus in which there is no marker and imprinted genes are not expressed after hematoxylin staining of the cells; the b is a red/brown mark in the nucleus after hematoxylin staining and imprinted genes are present in the cells. Cell nucleus; The c is a cell nucleus with two red/brown markers and imprinted gene deletion after hematoxylin staining of the cell; the d is a cell nucleus with more than two red/brown markers after hematoxylin staining. , nuclei with abnormal copy numbers of imprinted genes.
本发明中，所述苏木素染色后的标记选自但不限于红色或棕色，用其他颜色进行染色标记也可用于印迹基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。In the present invention, the hematoxylin-stained markers are selected from but not limited to red or brown. Staining markers with other colors can also be used to calculate the expression of imprinted genes, the expression of missing imprinted genes and the abnormal expression of imprinted gene copy numbers.
本发明中，将探针通过原位杂交，和Hemotoxy(苏木精)细胞核染色扩增信号，在40×或60×显微镜下，判断每一个细胞核内印记基因存在、印记基因缺失或拷贝数异常，通过计算印记基因缺失基因表达量和印记基因拷贝数异常的基因表达量来判定该样本的肿瘤良恶性程度。由于切片仅为10μm，所以在显微镜下所见细胞核大约有20％为不完整细胞核，也就是说有部分假阴性的可能性存在。In the present invention, the probe is amplified through in situ hybridization and Hemotoxy (hematoxylin) nuclear staining to amplify the signal. Under a 40× or 60× microscope, the presence, absence or copy number abnormality of the imprinted gene in each cell nucleus is determined. , by calculating the expression of imprinted gene deletion genes and the gene expression of imprinted gene copy number abnormalities to determine the benign and malignant degree of the tumor in the sample. Since the section is only 10 μm, approximately 20% of the cell nuclei seen under the microscope are incomplete nuclei, which means there is a possibility of some false negatives.
根据本发明，针对淋巴瘤的印记基因分级模型，所述印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级，通过每个探针在样本表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的八个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分别进行划分的五个不同的等级。According to the present invention, for the imprinted gene hierarchical model of lymphoma, the total expression amount of imprinted genes, the expression amount of imprinted gene deletion and the abnormal expression amount of imprinted gene copy number are divided into five different levels, and each probe is used to determine the highest expression level in the sample. Positive areas were counted for at least 1200 cells, and the expression of imprinted gene deletion, abnormal expression of imprinted gene copy number and total imprinted gene for eight imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 were counted. Expression levels are divided into five different levels.
根据本发明，所述针对Z1、Z11、Z13和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：According to the present invention, the five different levels divided into expression levels of imprinted gene deletion, imprinted gene copy number abnormal expression and total expression of Z1, Z11, Z13 and Z16 are:
0级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量小于16％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量小于1.5％或所述印记基因Z1、Z11、Z13和Z16的总表达量小于20％中的任意一种或至少两种的组合；Level 0: The imprinted gene deletion expression amount of imprinted genes Z1, Z11, Z13 and Z16 is less than 16%, the abnormal expression amount of imprinted gene copy number of imprinted genes Z1, Z11, Z13 and Z16 is less than 1.5% or the imprinted gene expression amount is less than 1.5%. The total expression level of genes Z1, Z11, Z13 and Z16 is less than 20% of any one or a combination of at least two of them;
I级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量为16-20％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量为1.5-2.5％或所述印记基因Z1、Z11、Z13和Z16的总表达量为20-30％中的任意一种或至少两种的组合；Level I: The imprinted gene deletion expression level of imprinted genes Z1, Z11, Z13 and Z16 is 16-20%, and the imprinted gene copy number abnormal expression level of imprinted genes Z1, Z11, Z13 and Z16 is 1.5-2.5%. Or the total expression level of the imprinted genes Z1, Z11, Z13 and Z16 is any one or a combination of at least two of 20-30%;
II级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量为2.5-5％或所述印记基因Z1、Z11、Z13和Z16的总表达量为30-40％中的任意一种或至少两种的组合；Level II: The imprinted gene deletion expression amount of imprinted genes Z1, Z11, Z13 and Z16 is 20-25%, and the imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z11, Z13 and Z16 is 2.5-5%. Or the total expression level of the imprinted genes Z1, Z11, Z13 and Z16 is any one or a combination of at least two of 30-40%;
III级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量为5-7％或所述印记基因Z1、Z11、Z13和Z16的总表达量为40-50％中的任意一种或至少两种的组合；Level III: The imprinted gene deletion expression level of imprinted genes Z1, Z11, Z13 and Z16 is 25-30%, and the imprinted gene copy number abnormal expression level of imprinted genes Z1, Z11, Z13 and Z16 is 5-7%. Or the total expression level of the imprinted genes Z1, Z11, Z13 and Z16 is any one or a combination of at least two of 40-50%;
IV级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量大于30％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量大于7％或所述印记基因Z1、Z11、Z13和Z16的总表达量大于50％中的任意一种或至少两种的组合；Level IV: The imprinted gene deletion expression amount of imprinted genes Z1, Z11, Z13 and Z16 is greater than 30%, the imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z11, Z13 and Z16 is greater than 7% or the imprinted gene expression amount is greater than 30%. The total expression level of genes Z1, Z11, Z13 and Z16 is greater than 50% of any one or a combination of at least two;
本发明中，所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。In the present invention, the imprinted gene deletion expression, imprinted gene copy number abnormal expression and total expression of imprinted genes Z1, Z11, Z13 and Z16 are independent of each other.
根据本发明，所述针对Z4、Z5、Z6和Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：According to the present invention, the five different levels divided into the expression amount of imprinted gene deletion, imprinted gene copy number abnormal expression amount and total expression amount of Z4, Z5, Z6 and Z8 are:
0级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量小于8％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量小于0.5％或所述印记基因Z4、Z5、Z6和Z8的总表达量小于15％中的任意一种或至少两种的组合；Level 0: The imprinted gene deletion expression amount of imprinted genes Z4, Z5, Z6 and Z8 is less than 8%, the abnormal expression amount of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is less than 0.5% or the imprinted gene expression amount is less than 8%. The total expression level of genes Z4, Z5, Z6 and Z8 is less than 15% of any one or a combination of at least two of them;
I级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量为8-15％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量为0.5-1.5％或所述印记基因Z4、Z5、Z6和Z8的总表达量为15-20％中的任意一种或至少两种的组合；Level I: The expression level of imprinted gene deletion of imprinted genes Z4, Z5, Z6 and Z8 is 8-15%, and the abnormal expression level of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is 0.5-1.5%. Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z8 is any one or a combination of at least two of 15-20%;
II级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量为15-20％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量为1.5-2.5％或所述印记基因Z4、Z5、Z6和Z8的总表达量为20-30％中的任意一种或至少两种的组合；Level II: The expression level of imprinted gene deletion of imprinted genes Z4, Z5, Z6 and Z8 is 15-20%, and the abnormal expression level of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is 1.5-2.5%. Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z8 is any one or a combination of at least two of 20-30%;
III级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量为20-25％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量为2.5-4％或所述印记基因Z4、Z5、Z6和Z8的总表达量为30-40％中的任意一种或至少两种的组合；Level III: The expression level of imprinted gene deletion of imprinted genes Z4, Z5, Z6 and Z8 is 20-25%, and the abnormal expression level of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is 2.5-4%. Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z8 is any one or a combination of at least two of 30-40%;
IV级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量大于25％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量大于4％或所述印记基因Z4、Z5、Z6和Z8的总表达量大于40％中的任意一种或至少两种的组合；Level IV: The imprinted gene deletion expression amount of imprinted genes Z4, Z5, Z6 and Z8 is greater than 25%, the imprinted gene Z4, Z5, Z6 and Z8 imprinted gene copy number abnormal expression amount is greater than 4% or the imprinted gene expression amount is greater than 25%. The total expression of genes Z4, Z5, Z6 and Z8 is greater than 40% of any one or a combination of at least two of them;
本发明中，所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。In the present invention, the imprinted gene deletion expression, imprinted gene copy number abnormal expression and total expression of the imprinted genes Z4, Z5, Z6 and Z8 are independent of each other.
根据本发明，针对淋巴转移癌的印记基因分级模型，所述印记基因拷贝数异常表达量分成三个不同的等级，通过每个探针在样本表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16的十五个印记基因的印记基因拷贝数异常表达量进行划分的三个不同的等级。According to the present invention, for the imprinted gene grading model of lymphatic metastasis cancer, the abnormal expression of imprinted gene copy number is divided into three different levels, and at least 1200 cells are counted in the most positive expression area of the sample through each probe, Three different classifications of abnormal expression levels of imprinted gene copy numbers for the fifteen imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 level.
根据本发明，所述针对Z1和Z16的印记基因拷贝数异常表达量划分的三个不同的等级为：According to the present invention, the three different levels for the abnormal expression of imprinted gene copy number of Z1 and Z16 are:
0级：所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于5％；Level 0: The abnormal expression amount of imprinted gene copy number of imprinted genes Z1 and Z16 is less than 5%;
I级：所述印记基因Z1和Z16的印记基因拷贝数异常表达量为5％-10％；Level I: The abnormal expression amount of imprinted gene copy number of imprinted genes Z1 and Z16 is 5%-10%;
II级：所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于10％；Level II: The abnormal expression of imprinted gene copy number of imprinted genes Z1 and Z16 is greater than 10%;
本发明中，所述印记基因Z1和Z16的印记基因拷贝数异常表达量是相互独立的。In the present invention, the abnormal expression levels of imprinted gene copy numbers of imprinted genes Z1 and Z16 are independent of each other.
根据本发明，所述针对Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量划分的三个不同的等级为：According to the present invention, the three different levels for the abnormal expression of imprinted gene copy number of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are:
0级：所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量小于2％；Level 0: The abnormal expression amount of imprinted gene copy number of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is less than 2%;
I级：所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量为2％-5％；Level I: The abnormal expression amount of imprinted gene copy number of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is 2%-5%;
II级：所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量大于5％；Level II: The abnormal expression of imprinted gene copy number of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is greater than 5%;
本发明中，所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量是相互独立的。In the present invention, the abnormal expression levels of imprinted gene copy numbers of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are independent of each other.
第三方面，本发明提供了一种检测淋巴瘤良恶性程度和癌症淋巴转移程度的装置，包括如下单元：In a third aspect, the present invention provides a device for detecting the benign and malignant degree of lymphoma and the degree of lymphatic metastasis of cancer, including the following units:
(1)取样单元：获取待测样本；(1) Sampling unit: obtain the sample to be tested;
(2)探针设计单元：根据印记基因序列设计特异性引物；(2) Probe design unit: Design specific primers based on imprinted gene sequences;
(3)检测单元：将步骤(2)的探针与待测样本进行原位杂交；(3) Detection unit: perform in situ hybridization between the probe of step (2) and the sample to be tested;
(4)分析单元：显微镜成像分析印记基因的表达情况；(4) Analysis unit: Microscope imaging analyzes the expression of imprinted genes;
其中，所述分析单元通过计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量，通过第一方面和/或第二方面所述的印记基因分级模型，从而通过印记基因缺失表达量和印记基因拷贝数异常表达量的等级来判断淋巴瘤的良恶性程度和癌症淋巴转移的程度。Wherein, the analysis unit calculates the total expression amount of imprinted genes, the expression amount of imprinted gene deletion and the abnormal expression amount of imprinted gene copy number, through the imprinted gene hierarchical model described in the first aspect and/or the second aspect, thereby through the imprinted gene The level of deletion expression and abnormal expression of imprinted gene copy number can be used to determine the benign and malignant degree of lymphoma and the degree of cancer lymphatic metastasis.
本发明中，所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记的细胞核，所述印记基因拷贝数异常为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记的细胞核，所述拷贝数异常是由于癌细胞异常地进行基因复制，导致这个基因表达时呈现为三倍体甚至更高的多倍体的情况。In the present invention, the imprinted gene deletion means that after hematoxylin staining of the cells, there are two red/brown labeled nuclei in the cell nucleus, and the imprinted gene copy number abnormality means that after the cells are hematoxylin stained, there are more than two nuclei in the cell nuclei. Red/brown marked cell nuclei, the copy number abnormality is due to abnormal gene replication in cancer cells, resulting in triploidy or even higher polyploidy when the gene is expressed.
本发明中，所述苏木素染色后的标记选自但不限于红色或棕色，用其他颜色进行染色标记也可用于印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。In the present invention, the hematoxylin-stained markers are selected from but not limited to red or brown. Staining markers with other colors can also be used to calculate the total expression of imprinted genes, the expression of missing imprinted genes and the abnormal expression of imprinted gene copy numbers. .
本发明所述检测装置是用于细胞和组织水平下早期直观地观察淋巴瘤和癌转移淋巴结的印记(迹)基因的变化从而判断肿瘤的良恶性程度和淋巴结中癌细胞转移的程度，为早期淋巴瘤患者和早期癌症转移患者提供最有利的治疗机会。The detection device of the present invention is used for early and intuitive observation of imprint (trace) gene changes in lymphoma and cancer metastasis lymph nodes at the cell and tissue level to determine the benign and malignant degree of tumors and the degree of cancer cell metastasis in lymph nodes. It is an early stage Lymphoma patients and patients with early-stage cancer metastases offer the most favorable treatment opportunities.
根据本发明，步骤(1)所述的待测样本来自于人的组织和/或细胞。According to the present invention, the sample to be tested in step (1) comes from human tissues and/or cells.
本发明中，所述待测样本只要RNA经过及时固定的处理都是可行的，本领域技术人员可以根据需要进行选择，在此不做特殊限定，本发明所述待测样本包括组织的石蜡切片和淋巴结穿刺活检样本中的任意一种或至少两种的组合。In the present invention, the sample to be tested is feasible as long as the RNA has been fixed in time. Those skilled in the art can make a selection according to needs. There is no special limitation here. The sample to be tested in the present invention includes paraffin sections of tissue. and any one or a combination of at least two of lymph node biopsy samples.
所述组织的石蜡切片具体操作步骤为获取人体肿瘤或淋巴结组织样本，及时用10％中性福尔马林固定，石蜡包埋，切成10μm厚，用带正电荷的玻片制成组织片子；因为只有10μm厚，因此显微镜下看见的有一部分为不完整的细胞核，所以会出现部分假阴性的基因缺失。The specific steps for paraffin sectioning of the tissue are to obtain human tumor or lymph node tissue samples, fix them in time with 10% neutral formalin, embed them in paraffin, cut them into 10 μm thick, and use positively charged glass slides to make tissue slices. ; Because it is only 10μm thick, part of what is seen under the microscope is incomplete cell nuclei, so there will be some false negative gene deletions.
所述淋巴结穿刺活检样本具体操作步骤为通过穿刺获取人体淋巴结细胞，及时用10％中性福尔马林固定即可。The specific operation steps of the lymph node puncture biopsy sample are to obtain human lymph node cells through puncture and fix them in time with 10% neutral formalin.
本发明中，由于淋巴结穿刺活检对病人伤害小，取样过程简单，相较于血液的循环特性，淋巴结穿刺活检还能定位，淋巴结穿刺活检作为实验样本有其特殊的优势。In the present invention, since lymph node puncture biopsy causes little harm to the patient and the sampling process is simple, compared with the circulation characteristics of blood, lymph node puncture biopsy can also be positioned. Lymph node puncture biopsy has its special advantages as an experimental sample.
优选地，所述待测样本为淋巴结穿刺活检样本。Preferably, the sample to be tested is a lymph node biopsy sample.
优选地，所述印记基因为Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16，所述印记基因Z1为Gnas，所述印记基因Z2为Igf2，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z9为Dlk1，所述印记基因Z10为Gatm，所述印记基因Z11为Grb10，所述印记基因Z12为Peg3，所述印记基因Z13为Sgce，所述印记基因Z14为Slc38a4，所述印记基因Z15为Diras3，所述印记基因Z16为Snrpn/Snurf。Preferably, the imprinted genes are Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, the imprinted gene Z1 is Gnas, and the imprinted gene Z2 is Igf2, the imprinted gene Z3 is Peg10, the imprinted gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, and the imprinted gene Z9 is Dlk1, the imprinted gene Z10 is Gatm, the imprinted gene Z11 is Grb10, the imprinted gene Z12 is Peg3, the imprinted gene Z13 is Sgce, the imprinted gene Z14 is Slc38a4, the imprinted gene Z15 is Diras3, The imprinted gene Z16 is Snrpn/Snurf.
本发明中，所述印记基因Z1(Gnas)，Z2(Igf2)，Z3(Peg10)，Z4(Igf2r)，Z5(Mest)，Z6(Plagl1)，Z8(Dcn)，Z9(Dlk1)，Z10(Gatm)，Z11(Grb10)，Z12(Peg3)，Z13(Sgce)，Z14(Slc38a4)，Z15(Diras3)，Z16(Snrpn/Snurf)在正常肿瘤细胞组织和淋巴结内有不同程度的表达，在发生恶性病变时，表达量和印记状态都会发生明显变化。In the present invention, the imprinted genes Z1 (Gnas), Z2 (Igf2), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z9 (Dlk1), Z10 ( Gatm), Z11 (Grb10), Z12 (Peg3), Z13 (Sgce), Z14 (Slc38a4), Z15 (Diras3), and Z16 (Snrpn/Snurf) are expressed to varying degrees in normal tumor cell tissues and lymph nodes. In malignant lesions, the expression level and imprinting status will change significantly.
本发明中，所述设计探针是根据印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16，即Gnas，Igf2，Peg10，Igf2r，Mest，Plagl1，Dcn，Dlk1，Gatm，Grb10，Peg3，Sgce，Slc38a4，Diras3和Snrpn/Snurf进行设计的，具体在每个基因的内含子内选择一段序列作为探针，具体的探针由Advanced Cell Diagnostics公司设计。In the present invention, the designed probe is based on imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, namely Gnas, Igf2, Peg10, Igf2r, Mest, Plagl1, Dcn, Dlk1, Gatm, Grb10, Peg3, Sgce, Slc38a4, Diras3 and Snrpn/Snurf were designed. Specifically, a sequence was selected as a probe within the intron of each gene. The specific probe Designed by Advanced Cell Diagnostics.
优选地，所述原位杂交采用RNAscope原位杂交方法。Preferably, the in situ hybridization adopts RNAscope in situ hybridization method.
优选地，所述RNAscope原位杂交方法使用单通道或多通道的呈色试剂盒或者单通道或多通道的荧光试剂盒，优选为单通道红色/棕色呈色试剂盒或多通道的荧光试剂盒。Preferably, the RNAscope in situ hybridization method uses a single-channel or multi-channel coloring kit or a single-channel or multi-channel fluorescence kit, preferably a single-channel red/brown coloring kit or a multi-channel fluorescence kit. .
本发明中，所述多通道呈色试剂盒或多通道荧光试剂盒包括两通道或两通道以上的呈色试剂盒或荧光试剂盒，所述两通道的呈色试剂盒或多通道的荧光试剂盒可以使用两个印记基因探针或印记基因和其他基因的联合表达甚至多个印记基因和非印记基因的综合表达。In the present invention, the multi-channel coloring kit or multi-channel fluorescence kit includes a two-channel or more than two-channel coloring kit or a fluorescence kit, and the two-channel coloring kit or multi-channel fluorescence reagent The cassette can use two imprinted gene probes or the combined expression of an imprinted gene and other genes or even the combined expression of multiple imprinted genes and non-imprinted genes.
根据本发明，所述模型中的计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：According to the present invention, the formula for calculating the total expression of imprinted genes, the expression of missing imprinted genes and the abnormal expression of imprinted gene copy numbers in the model is as follows:
总表达量＝(b+c+d)/(a+b+c+d)×100％；Total expression amount=(b+c+d)/(a+b+c+d)×100%;
正常印记基因表达量＝b/(b+c+d)×100％；Normal imprinted gene expression = b/(b+c+d)×100%;
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；Imprinted gene deletion gene expression level (LOI) = c/(b+c+d)×100%;
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；Gene expression amount with abnormal imprinted gene copy number (CNV) = d/(b+c+d)×100%;
其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。Wherein, the a refers to a cell nucleus in which after hematoxylin staining, there is no marker in the nucleus and imprinted genes are not expressed; the b refers to a red/brown mark in the nucleus after hematoxylin staining in the cells and the presence of imprinted genes. Cell nucleus; The c is a cell nucleus with two red/brown markers and imprinted gene missing after hematoxylin staining of the cell; the d is a cell nucleus with more than two red/brown markers after hematoxylin staining. , nuclei with abnormal copy numbers of imprinted genes.
本发明中，所述苏木素染色后的标记选自但不限于红色或棕色，用其他颜色进行染色标记也可用于印迹基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。In the present invention, the hematoxylin-stained markers are selected from but not limited to red or brown. Staining markers with other colors can also be used to calculate the expression of imprinted genes, the expression of missing imprinted genes and the abnormal expression of imprinted gene copy numbers.
本发明中，将探针通过原位杂交，和Hemotoxy(苏木精)细胞核染色扩增信号，在40×或60×显微镜下，判断每一个细胞核内印记基因存在、印记基因缺失或拷贝数异常，通过计算印记基因总表达量、印记基因缺失基因表达量和印记基因拷贝数异常的基因表达量来判定该样本的肿瘤良恶性程度和淋巴结中癌细胞转移的程度。由于切片仅为10微米，所以在显微镜下所见细胞核大约有20％为不完整细胞核，也就是说有部分假阴性的可能性存在。In the present invention, the probe is amplified through in situ hybridization and Hemotoxy (hematoxylin) nuclear staining to amplify the signal. Under a 40× or 60× microscope, the presence, absence or copy number abnormality of the imprinted gene in each cell nucleus is determined. , by calculating the total expression of imprinted genes, the expression of imprinted genes missing genes and the expression of genes with abnormal copy number of imprinted genes to determine the benign or malignant degree of the tumor and the degree of cancer cell metastasis in the lymph nodes. Since the section is only 10 microns, approximately 20% of the nuclei seen under the microscope are incomplete nuclei, which means there is a possibility of some false negatives.
根据本发明，针对淋巴瘤的印记基因分级模型，所述印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量分成五个不同的等级。According to the present invention, for the imprinted gene hierarchical model of lymphoma, the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount are divided into five different levels.
所述五个不同的等级为在样本每个探针表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的八个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量分别进行划分。The five different levels are based on counting at least 1200 cells in the most positive area of expression of each probe in the sample, targeting the eight imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16. The expression amount of gene deletion, abnormal expression amount of imprinted gene copy number and total expression amount were divided respectively.
所述针对Z1、Z11、Z13和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：The five different levels divided into expression levels of imprinted gene deletion, imprinted gene copy number abnormal expression and total expression of Z1, Z11, Z13 and Z16 are as follows:
0级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量小于16％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量小于1.5％或所述印记基因Z1、Z11、Z13和Z16的总表达量小于20％中的任意一种或至少两种的组合；Level 0: The imprinted gene deletion expression amount of imprinted genes Z1, Z11, Z13 and Z16 is less than 16%, the abnormal expression amount of imprinted gene copy number of imprinted genes Z1, Z11, Z13 and Z16 is less than 1.5% or the imprinted gene expression amount is less than 1.5%. The total expression level of genes Z1, Z11, Z13 and Z16 is less than 20% of any one or a combination of at least two of them;
I级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量为16-20％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量为1.5-2.5％或所述印记基因Z1、Z11、Z13和Z16的总表达量为20-30％中的任意一种或至少两种的组合；Level I: The imprinted gene deletion expression level of imprinted genes Z1, Z11, Z13 and Z16 is 16-20%, and the imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z11, Z13 and Z16 is 1.5-2.5%. Or the total expression level of the imprinted genes Z1, Z11, Z13 and Z16 is any one or a combination of at least two of 20-30%;
II级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量为2.5-5％或所述印记基因Z1、Z11、Z13和Z16的总表达量为30-40％中的任意一种或至少两种的组合；Level II: The imprinted gene deletion expression amount of imprinted genes Z1, Z11, Z13 and Z16 is 20-25%, and the imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z11, Z13 and Z16 is 2.5-5%. Or the total expression level of the imprinted genes Z1, Z11, Z13 and Z16 is any one or a combination of at least two of 30-40%;
III级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量为5-7％或所述印记基因Z1、Z11、Z13和Z16的总表达量为40-50％中的任意一种或至少两种的组合；Level III: The imprinted gene deletion expression level of imprinted genes Z1, Z11, Z13 and Z16 is 25-30%, and the imprinted gene copy number abnormal expression level of imprinted genes Z1, Z11, Z13 and Z16 is 5-7%. Or the total expression level of the imprinted genes Z1, Z11, Z13 and Z16 is any one or a combination of at least two of 40-50%;
IV级：所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量大于30％、所述印记基因Z1、Z11、Z13和Z16的印记基因拷贝数异常表达量大于7％或所述印记基因Z1、Z11、Z13和Z16的总表达量大于50％中的任意一种或至少两种的组合；Level IV: The imprinted gene deletion expression amount of imprinted genes Z1, Z11, Z13 and Z16 is greater than 30%, the imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z11, Z13 and Z16 is greater than 7% or the imprinted gene expression amount is greater than 30%. The total expression level of genes Z1, Z11, Z13 and Z16 is greater than 50% of any one or a combination of at least two;
本发明中，所述印记基因Z1、Z11、Z13和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。In the present invention, the imprinted gene deletion expression, imprinted gene copy number abnormal expression and total expression of imprinted genes Z1, Z11, Z13 and Z16 are independent of each other.
所述针对Z4、Z5、Z6和Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：The five different levels divided into the expression levels of imprinted gene deletions, imprinted gene copy number abnormal expression levels and total expression levels of Z4, Z5, Z6 and Z8 are as follows:
0级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量小于8％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量小于0.5％或所述印记基因Z4、Z5、Z6和Z8的总表达量小于15％中的任意一种或至少两种的组合；Level 0: The imprinted gene deletion expression amount of imprinted genes Z4, Z5, Z6 and Z8 is less than 8%, the abnormal expression amount of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is less than 0.5% or the imprinted gene expression amount is less than 8%. The total expression level of genes Z4, Z5, Z6 and Z8 is less than 15% of any one or a combination of at least two of them;
I级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量为8-15％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量为0.5-1.5％或所述印记基因Z4、Z5、Z6和Z8的总表达量为15-20％中的任意一种或至少两种的组合；Level I: The expression level of imprinted gene deletion of imprinted genes Z4, Z5, Z6 and Z8 is 8-15%, and the abnormal expression level of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is 0.5-1.5%. Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z8 is any one or a combination of at least two of 15-20%;
II级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量为15-20％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量为1.5-2.5％或所述印记基因Z4、Z5、Z6和Z8的总表达量为20-30％中的任意一种或至少两种的组合；Level II: The expression level of imprinted gene deletion of imprinted genes Z4, Z5, Z6 and Z8 is 15-20%, and the abnormal expression level of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is 1.5-2.5%. Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z8 is any one or a combination of at least two of 20-30%;
III级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量为20-25％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量为2.5-4％或所述印记基因Z4、Z5、Z6和Z8的总表达量为30-40％中的任意一种或至少两种的组合；Level III: The expression level of imprinted gene deletion of imprinted genes Z4, Z5, Z6 and Z8 is 20-25%, and the abnormal expression level of imprinted gene copy number of imprinted genes Z4, Z5, Z6 and Z8 is 2.5-4%. Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z8 is any one or a combination of at least two of 30-40%;
IV级：所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量大于25％、所述印记基因Z4、Z5、Z6和Z8的印记基因拷贝数异常表达量大于4％或所述印记基因Z4、Z5、Z6和Z8的总表达量大于40％中的任意一种或至少两种的组合；Level IV: The imprinted gene deletion expression amount of imprinted genes Z4, Z5, Z6 and Z8 is greater than 25%, the imprinted gene Z4, Z5, Z6 and Z8 imprinted gene copy number abnormal expression amount is greater than 4% or the imprinted gene expression amount is greater than 25%. The total expression of genes Z4, Z5, Z6 and Z8 is greater than 40% of any one or a combination of at least two of them;
本发明中，所述印记基因Z4、Z5、Z6和Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。In the present invention, the imprinted gene deletion expression, imprinted gene copy number abnormal expression and total expression of the imprinted genes Z4, Z5, Z6 and Z8 are independent of each other.
优选地，所述判断淋巴瘤的良恶性程度分为良性肿瘤、恶性潜能淋巴瘤、早期恶性淋巴瘤、中期恶性淋巴瘤和晚期恶性淋巴瘤；Preferably, the judged benign and malignant degree of lymphoma is divided into benign tumors, malignant potential lymphomas, early malignant lymphomas, intermediate malignant lymphomas and late malignant lymphomas;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression levels of imprinted gene deletion and imprinted gene copy number abnormal expression of imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 are less than level I. or the imprinted gene deletion expression level of no more than 1 imprinted gene Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I and the imprinted gene Z1, Z4, Z5, Z6, Z8, Z11, If the abnormal expression of imprinted gene copy number of no more than one imprinted gene in Z13 and Z16 is grade I, it is a benign tumor;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则为恶性潜能淋巴瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I, and the imprinted gene deletion expression level is level I. The abnormal expression amount of imprinted gene copy number of at least 2 imprinted genes of genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I or the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and The imprinted gene deletion expression level of no more than 1 imprinted gene in Z16 is level II and the imprinted gene copy number abnormal expression of no more than 1 imprinted gene in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 If the amount is any one of grade II, it is lymphoma with malignant potential;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期恶性淋巴瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level II, and the imprinted gene deletion expression level is level II. The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level II or the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and The imprinted gene deletion expression level of no more than 1 imprinted gene in Z16 is level III and the imprinted gene copy number abnormal expression of no more than 1 imprinted gene in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 If the amount is any one of grade III, it is early malignant lymphoma;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期恶性淋巴瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level III, and the imprinted gene deletion expression level is level III. The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level III or the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and The imprinted gene deletion expression level of no more than 1 imprinted gene in Z16 is level IV and the imprinted gene copy number abnormal expression of no more than 1 imprinted gene in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 If the amount is any one of grade IV, it is an intermediate malignant lymphoma;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶性淋巴瘤。Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level IV or the imprinted gene If the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes among Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade IV, it is an advanced malignant lymphoma.
根据本发明，针对淋巴转移癌的印记基因分级模型，所述印记基因拷贝数异常表达量分成三个不同的等级。According to the present invention, for the imprinted gene hierarchical model of lymphatic metastasis cancer, the abnormal expression amount of imprinted gene copy number is divided into three different levels.
所述三个不同的等级为在样本表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16的十五个印记基因的印记基因拷贝数异常表达量分别进行划分。The three different levels are to count at least 1200 cells in the most positive expression area of the sample, targeting Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, The abnormal expression amounts of imprinted gene copy numbers of the fifteen imprinted genes of Z15 and Z16 were divided respectively.
所述针对Z1和Z16的印记基因拷贝数异常表达量划分的三个不同的等级为：The three different levels of abnormal expression levels of imprinted gene copy numbers of Z1 and Z16 are:
0级：所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于5％；Level 0: The abnormal expression amount of imprinted gene copy number of imprinted genes Z1 and Z16 is less than 5%;
I级：所述印记基因Z1和Z16的印记基因拷贝数异常表达量为5％-10％；Level I: The abnormal expression amount of imprinted gene copy number of imprinted genes Z1 and Z16 is 5%-10%;
II级：所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于10％；Level II: The abnormal expression of imprinted gene copy number of imprinted genes Z1 and Z16 is greater than 10%;
本发明中，所述印记基因Z1和Z16的印记基因拷贝数异常表达量是相互独立的。In the present invention, the abnormal expression levels of imprinted gene copy numbers of imprinted genes Z1 and Z16 are independent of each other.
所述针对Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量划分的三个不同的等级为：The three different levels of abnormal expression levels of imprinted gene copy numbers of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are:
0级：所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量小于2％；Level 0: The abnormal expression amount of imprinted gene copy number of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is less than 2%;
I级：所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量为2％-5％；Level I: The abnormal expression amount of imprinted gene copy number of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is 2%-5%;
II级：所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量大于5％；Level II: The abnormal expression of imprinted gene copy number of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is greater than 5%;
本发明中，所述印记基因Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14和Z15的印记基因拷贝数异常表达量是相互独立的。In the present invention, the abnormal expression levels of imprinted gene copy numbers of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are independent of each other.
优选地，判断淋巴转移癌的癌细胞转移程度分为未转移、轻度转移和重度转移。Preferably, the degree of cancer cell metastasis for lymphatic metastasis cancer is judged into non-metastasis, mild metastasis and severe metastasis.
优选地，所述判断淋巴转移癌的癌细胞转移程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16的印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为未转移；Preferably, the result of judging the degree of cancer cell metastasis of lymphatic metastasis cancer is the imprint of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal expression of gene copy number is less than level I or no more than 1 imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 If the abnormal expression level of imprinted gene copy number is level I, it means it has not been transferred;
优选地，所述判断淋巴转移癌的癌细胞转移程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级，则为轻度转移；Preferably, the result for judging the degree of cancer cell metastasis of lymphatic metastasis cancer is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal expression level of imprinted gene copy number for at least 2 imprinted genes is level I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 If the abnormal expression level of imprinted gene copy number is no more than 1 imprinted gene and is grade II, it is considered as mild metastasis;
优选地，所述判断淋巴转移癌的癌细胞转移程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的至少2个印记基因的印记基因拷贝数异常表达量为II级，则为重度转移。Preferably, the result for judging the degree of cancer cell metastasis of lymphatic metastasis cancer is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 If the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes is level II, it is considered as severe metastasis.
第四方面，本发明提供一种如第一方面和/或第二方面所述的模型或如第三方面所述的装置用于制备淋巴瘤和/或淋巴转移癌的检测和/或治疗的药物或试剂盒。In a fourth aspect, the present invention provides a model as described in the first aspect and/or the second aspect or a device as described in the third aspect for preparing a method for detecting and/or treating lymphoma and/or lymphatic metastasis cancer. Medications or kits.
优选地，判断淋巴瘤的良恶性程度分为良性肿瘤、恶性潜能淋巴瘤、早期恶性淋巴瘤、中期恶性淋巴瘤和晚期恶性淋巴瘤；Preferably, the benign and malignant degree of lymphoma is judged into benign tumors, malignant potential lymphomas, early malignant lymphomas, intermediate malignant lymphomas and late malignant lymphomas;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression levels of imprinted gene deletion and imprinted gene copy number abnormal expression of imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 are less than level I. or the imprinted gene deletion expression level of no more than 1 imprinted gene Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I and the imprinted gene Z1, Z4, Z5, Z6, Z8, Z11, If the abnormal expression of imprinted gene copy number of no more than one imprinted gene in Z13 and Z16 is grade I, it is a benign tumor;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则判断为恶性潜能淋巴瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I, and the imprinted gene deletion expression level is level I. The abnormal expression amount of imprinted gene copy number of at least 2 imprinted genes of genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I or the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and No more than 1 imprinted gene in Z16 has imprinted gene deletion expression level II and no more than 1 imprinted gene in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 has abnormal expression of imprinted gene copy number If the amount is any one of grade II, it is judged to be lymphoma with malignant potential;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期恶性淋巴瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level II, and the imprinted gene deletion expression level is level II. The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level II or the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and The imprinted gene deletion expression level of no more than 1 imprinted gene in Z16 is level III and the imprinted gene copy number abnormal expression of no more than 1 imprinted gene in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 If the amount is any one of grade III, it is early malignant lymphoma;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期恶性淋巴瘤；Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level III, and the imprinted gene deletion expression level is level III. The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level III or the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and The imprinted gene deletion expression level of no more than 1 imprinted gene in Z16 is level IV and the imprinted gene copy number abnormal expression of no more than 1 imprinted gene in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 If the amount is any one of grade IV, it is an intermediate malignant lymphoma;
优选地，所述判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶性淋巴瘤。Preferably, the result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least two of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level IV or the imprinted gene If the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes among Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade IV, it is an advanced malignant lymphoma.
优选地，判断淋巴转移癌中的转移程度分为未转移、轻度转移和重度转移；Preferably, the degree of metastasis in lymphatic metastasis cancer is judged into non-metastasis, mild metastasis and severe metastasis;
优选地，所述判断淋巴转移癌中的转移程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16的印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为未转移；Preferably, the result for judging the degree of metastasis in lymphatic metastasis cancer is the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal copy number expressions are all less than level I or no more than 1 imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 If the abnormal expression level of imprinted gene copy number is level I, it means it has not been transferred;
优选地，所述判断淋巴转移癌中的转移程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级，则为轻度转移；Preferably, the result of judging the degree of metastasis in lymphatic metastasis cancer is at least one of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16. The abnormal expression level of imprinted gene copy number of 2 imprinted genes is level I or different among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16. If the abnormal expression level of imprinted gene copy number is level II for more than one imprinted gene, it is considered as mild metastasis;
优选地，所述判断淋巴转移癌中的转移程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的至少2个印记基因的印记基因拷贝数异常表达量为II级，则为重度转移。Preferably, the result of judging the degree of metastasis in lymphatic metastasis cancer is at least one of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16. The abnormal expression level of imprinted gene copy number of two imprinted genes is level II, which indicates severe metastasis.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)本发明所述检测模型和装置，以直观的方法表现了印记缺失在淋巴瘤和癌症淋巴转移病人的样本上的表现，通过对印记基因原位标记的方法，客观，直观，早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为淋巴瘤和癌症淋巴转移的诊断做出巨大贡献；(1) The detection model and device of the present invention express the performance of imprinting loss in samples of patients with lymphoma and cancer lymphatic metastasis in an intuitive way. Through the method of in-situ labeling of imprinting genes, it is objective, intuitive, and early. It can accurately detect changes in imprinted (trace) genes and provide a quantitative model, making a huge contribution to the diagnosis of lymphoma and cancer lymphatic metastasis;
(2)本发明检测装置，可以在淋巴瘤病人手术前通过淋巴结穿刺活检细胞得出淋巴瘤良恶性程度的判断，从而为手术及精准治疗提供依据，这是细胞分子领域诊断淋巴瘤的革命性突破；(2) The detection device of the present invention can determine the benign and malignant degree of lymphoma through lymph node biopsy cells before surgery in lymphoma patients, thereby providing a basis for surgery and precise treatment. This is revolutionary in the diagnosis of lymphoma in the field of cell molecules. breakthrough;
(3)本发明可以准确区分良性淋巴增生和早期恶性淋巴癌，通过印记基因的组合检测对淋巴瘤的恶性程度进行明确地分级，极大地提高了对恶性淋巴瘤的早期、明确诊断，取材过程简单，用在早期普查和癌症术后随访，尤其是对于疑似复发病人的跟踪随访，可以争取时间，为挽救病人生命做出重大贡献；(3) The present invention can accurately distinguish benign lymphoid hyperplasia and early malignant lymphoma, and clearly grade the malignancy of lymphoma through the combined detection of imprinted genes, which greatly improves the early and clear diagnosis of malignant lymphoma and the material collection process. Simple, it can be used for early screening and post-cancer follow-up, especially for patients with suspected recurrence, which can buy time and make a significant contribution to saving the patient's life;
(4)本发明检测装置，可以在癌症病人手术前通过淋巴结穿刺得出癌细胞是否已经发生淋巴转移并判断转移范围，帮助医生确定手术切除和淋巴结清扫的范围，以及术后治疗方案的选择，极大减少术后复发；(4) The detection device of the present invention can determine whether cancer cells have metastasized to lymph nodes and determine the extent of metastasis through lymph node puncture before surgery, helping doctors determine the scope of surgical resection and lymph node dissection, as well as the selection of postoperative treatment plans. Greatly reduce postoperative recurrence;
(5)本发明检测方法区别于免疫组化方法，减少了假阳性和其他负面作用，不仅如此，通过发现的淋巴瘤相关印记基因缺失位点的致该基因沉默、剔除、重排的靶向药物或技术方法，可用于指导后期的治疗和用药。(5) The detection method of the present invention is different from the immunohistochemical method in that it reduces false positives and other negative effects. Not only that, it also targets the silencing, deletion, and rearrangement of the discovered lymphoma-related imprinted gene deletion sites. Drugs or technical methods can be used to guide later treatment and medication.
附图说明Description of drawings
图1是本发明苏木素染色细胞核的恶性淋巴瘤的病理切片，其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常；Figure 1 is a pathological section of malignant lymphoma with hematoxylin-stained cell nuclei according to the present invention. The a shows that after the cells were hematoxylin-stained, there was no marker in the nucleus and the imprinted genes were not expressed; the b shows that the cells were hematoxylin-stained. , there is a red/brown mark in the nucleus, and the imprinted gene is present; the c means that after the cells are stained with hematoxylin, there are two red/brown marks in the nucleus, and the imprinted gene is missing; the d means that after the cells are stained with hematoxylin, There are more than two red/brown marks in the nucleus, and the copy number of imprinted genes is abnormal;
图2(a)为0级淋巴瘤的病理切片中8个基因的表达状态，图2(b)为I级淋巴瘤的病理切片中8个基因的表达状态，图2(c)为II级淋巴瘤的病理切片中8个基因的表达状态，图2(d)为III级淋巴瘤的病理切片中8个基因的表达状态，图2(e)为IV级淋巴瘤的病理切片中8个基因的表达状态；Figure 2(a) shows the expression status of 8 genes in the pathological sections of grade 0 lymphoma. Figure 2(b) shows the expression status of 8 genes in the pathological sections of grade I lymphoma. Figure 2(c) shows the expression status of 8 genes in the pathological sections of grade II lymphoma. The expression status of 8 genes in the pathological sections of lymphoma. Figure 2(d) shows the expression status of 8 genes in the pathological sections of grade III lymphoma. Figure 2(e) shows the expression status of 8 genes in the pathological sections of grade IV lymphoma. The expression status of genes;
图3(a)为印记基因Z1、Z8和Z16对淋巴瘤的印记缺失的强度，图3(b)为印记基因Z1、Z8和Z16对淋巴瘤的拷贝数异常的强度，图3(c)为印记基因Z1、Z8和Z16对淋巴瘤的总表达量的强度，图3(d)为印记基因Z4、Z5、Z6、Z11和Z13对淋巴瘤的印记缺失的强度，图3(e)为印记基因Z4、Z5、Z6、Z11和Z13对淋巴瘤的拷贝数异常的强度，图3(f)为印记基因Z4、Z5、Z6、Z11和Z13对淋巴瘤的总表达量的强度，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量，TE为印记基因总表达量；Figure 3(a) shows the intensity of imprinted genes Z1, Z8 and Z16 on imprinting loss in lymphoma. Figure 3(b) shows the intensity of imprinted genes Z1, Z8 and Z16 on lymphoma copy number abnormalities. Figure 3(c) is the intensity of the total expression of imprinted genes Z1, Z8 and Z16 on lymphoma, Figure 3(d) is the intensity of the imprinting loss of imprinted genes Z4, Z5, Z6, Z11 and Z13 on lymphoma, Figure 3(e) is The intensity of copy number abnormalities of imprinted genes Z4, Z5, Z6, Z11 and Z13 in lymphoma. Figure 3(f) shows the intensity of the total expression of imprinted genes Z4, Z5, Z6, Z11 and Z13 in lymphoma. Among them, LOI is the expression level of genes with missing imprinted genes, CNV is the expression level of genes with abnormal copy number of imprinted genes, and TE is the total expression level of imprinted genes;
图4(a)为印记基因Z1印记缺失、拷贝数异常和总表达量的强度，图4(b)为印记基因Z8印记缺失、拷贝数异常和总表达量的强度，图4(c)为印记基因Z16印记缺失、拷贝数异常和总表达量的强度，图4(d)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度，图4(e)为印记基因Z5印记缺失、拷贝数异常和总表达量的强度，图4(f)为印记基因Z6印记缺失、拷贝数异常和总表达量的强度，图4(g)为印记基因Z11印记缺失、拷贝数异常和总表达量的强度，图4(h)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量，TE为印记基因总表达量；Figure 4(a) shows the intensity of imprint loss, copy number abnormality and total expression of imprinted gene Z1. Figure 4(b) shows the intensity of imprint loss, copy number abnormality and total expression of imprinted gene Z8. Figure 4(c) shows The intensity of imprint loss, copy number abnormality and total expression of imprinted gene Z16. Figure 4(d) shows the intensity of imprint loss, copy number abnormality and total expression of imprinted gene Z4. Figure 4(e) shows the imprint loss, copy number abnormality and total expression of imprinted gene Z5. The intensity of copy number abnormality and total expression. Figure 4(f) shows the intensity of imprint loss, copy number abnormality and total expression of imprinted gene Z6. Figure 4(g) shows the imprint loss, copy number abnormality and total expression of imprinted gene Z11. Figure 4(h) shows the intensity of imprinting loss, copy number abnormality and total expression of imprinted gene Z13, where LOI is the gene expression level of imprinted gene deletion, CNV is the gene expression level of imprinted gene copy number abnormality, TE is the total expression amount of imprinted genes;
图5(a)为印记基因Z1应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(b)为印记基因Z8应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(c)为印记基因Z16应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(d)为印记基因Z4应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(e)为印记基因Z5应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(f)为印记基因Z6应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(g)为印记基因Z11应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图5(h)为印记基因Z13应用于77例淋巴瘤病理切片中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量，TE为印记基因总表达量；Figure 5(a) shows the distribution range and grading standards of imprinted gene Z1 applied to 77 cases of lymphoma pathological sections, copy number abnormalities and total expression. Figure 5(b) shows the application of imprinted gene Z8 to 77 cases of lymphoma. The distribution range and grading standard of imprinting loss, copy number abnormality and total expression in tumor pathological sections. Figure 5(c) shows the imprinting loss, copy number abnormality and total expression of imprinting gene Z16 in pathological sections of 77 cases of lymphoma. The distribution range and grading standards of the amount. Figure 5(d) shows the distribution range and grading standards of the imprinting gene Z4 applied to the pathological sections of 77 cases of lymphoma. Figure 5(e) shows the distribution range and grading standards of imprinting loss, copy number abnormality and total expression. Imprinted gene Z5 was applied to the pathological sections of 77 cases of lymphoma. The distribution range and grading standard of imprinting loss, copy number abnormality and total expression. Figure 5(f) shows the application of imprinted gene Z6 to the pathological sections of 77 cases of lymphoma. Imprinted The distribution range and grading standards of deletion, copy number abnormality and total expression. Figure 5(g) shows the distribution range and grading of imprinting deletion, copy number abnormality and total expression when the imprinted gene Z11 was applied to the pathological sections of 77 cases of lymphoma. Standard, Figure 5(h) shows the distribution range and grading standard of imprinting gene Z13 applied to 77 cases of lymphoma pathological sections, imprinting loss, copy number abnormality and total expression, where LOI is the expression level of imprinted gene deletion gene, CNV is the expression level of genes with abnormal copy number of imprinted genes, TE is the total expression level of imprinted genes;
图6(a)为未转移的乳腺癌附近淋巴结的穿刺细胞样本中15个基因的表达状态，图6(b)为轻度转移的乳腺癌附近淋巴结的穿刺细胞样本中15个基因的表达状态，图6(c)为重度转移的乳腺癌附近淋巴结的穿刺细胞样本中15个基因的表达状态；Figure 6(a) shows the expression status of 15 genes in the puncture cell samples of lymph nodes near non-metastatic breast cancer. Figure 6(b) shows the expression status of 15 genes in the puncture cell samples of lymph nodes near mildly metastasized breast cancer. , Figure 6(c) shows the expression status of 15 genes in puncture cell samples of lymph nodes near severely metastasized breast cancer;
图7(a)为印记基因Z1应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(b)为印记基因Z2应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(c)为印记基因Z3应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(d)为印记基因Z4应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(e)为印记基因Z5应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(f)为印记基因Z6应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(g)为印记基因Z8应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(h)为印记基因Z9应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(i)为印记基因Z10应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(j)为印记基因Z11应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(k)为印记基因Z12应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(l)为印记基因Z13应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(m)为印记基因Z14应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(n)为印记基因Z15应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，图7(o)为印记基因Z16应用于42例癌症患者淋巴结穿刺样本中，拷贝数异常的分布范围和分级标准，其中，CNV为印记基因拷贝数异常的基因表达量。Figure 7(a) shows the distribution range and grading standard of copy number abnormalities when imprinted gene Z1 was applied to lymph node puncture samples from 42 cancer patients. Figure 7(b) shows imprinted gene Z2 applied to lymph node puncture samples from 42 cancer patients. The distribution range and grading standards of copy number abnormalities. Figure 7(c) shows the application of imprinted gene Z3 in lymph node puncture samples of 42 cancer patients. The distribution range and grading standards of copy number abnormalities. Figure 7(d) shows the application of imprinted gene Z4. Figure 7(e) shows the distribution range and grading standards of copy number abnormalities in lymph node puncture samples of 42 cancer patients. Figure 7(e) shows the distribution range and grading standards of copy number abnormalities when imprinted gene Z5 was applied to lymph node puncture samples of 42 cancer patients. Figure 7(f) shows the distribution range and grading standard of copy number abnormalities when imprinted gene Z6 was applied to lymph node puncture samples from 42 cancer patients. Figure 7(g) shows imprinted gene Z8 was applied to lymph node puncture samples from 42 cancer patients. The distribution range and grading standards of copy number abnormalities. Figure 7(h) shows the application of imprinted gene Z9 in lymph node puncture samples of 42 cancer patients. The distribution range and grading standards of copy number abnormalities. Figure 7(i) shows the application of imprinted gene Z10. Figure 7(j) shows the distribution range and grading standards of copy number abnormalities in lymph node puncture samples of 42 cancer patients. Figure 7(j) shows the distribution range and grading standards of copy number abnormalities when imprinted gene Z11 was applied to lymph node puncture samples of 42 cancer patients. Figure 7(k) shows the distribution range and grading standard of copy number abnormalities when imprinted gene Z12 was applied to lymph node puncture samples from 42 cancer patients. Figure 7(l) shows imprinted gene Z13 applied to lymph node puncture samples from 42 cancer patients. The distribution range and grading standards of copy number abnormalities. Figure 7(m) shows the application of imprinted gene Z14 in lymph node puncture samples of 42 cancer patients. The distribution range and grading standards of copy number abnormalities. Figure 7(n) shows the application of imprinted gene Z15. The distribution range and grading standards of copy number abnormalities in the lymph node puncture samples of 42 cancer patients. Figure 7(o) shows the distribution range and grading standards of copy number abnormalities when the imprinted gene Z16 was applied to the lymph node puncture samples of 42 cancer patients. Among them, CNV is the gene expression level with abnormal copy number of imprinted genes.
具体实施方式Detailed ways
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。In order to further elucidate the technical means adopted by the present invention and its effects, the technical solutions of the present invention will be further described below with reference to the drawings and through specific implementations, but the present invention is not limited to the scope of the embodiments.
实施例1恶性淋巴瘤的印记基因分析Example 1 Analysis of imprinted genes in malignant lymphoma
所述的印记基因的检测方法，包括如下步骤：The method for detecting imprinted genes includes the following steps:
(1)获取恶性淋巴瘤的组织细胞切片(10微米)，放入10％中性福尔马林溶液中进行固定，以防RNA降解，固定时间为24小时，石蜡包埋(FFPE)，所述玻片需要用正电荷脱载玻片，所述切片在40℃烤箱烘烤3h以上；(1) Obtain tissue cell sections (10 microns) of malignant lymphoma and fix them in 10% neutral formalin solution to prevent RNA degradation. The fixation time is 24 hours and paraffin embedded (FFPE). The slides need to be desoldered with positive charge, and the slices are baked in a 40°C oven for more than 3 hours;
(2)按照RNASCope的样品处理方法进行脱蜡处理，封闭样本中内源性过氧化物酶活性，增强通透性并暴露出RNA分子；(2) Perform dewaxing according to the RNASCope sample processing method to block endogenous peroxidase activity in the sample, enhance permeability and expose RNA molecules;
(3)设计探针：根据印记基因序列设计特异性引物；(3) Design probes: Design specific primers based on imprinted gene sequences;
所述设计探针是根据印记基因Z1(Gnas)、Z4(Igf2r)、Z5(Mest)、Z6(Plagl1)、Z8(Dcn)、Z11(Grb10)、Z13(Sgce)和Z16(Snrpn/Snurf)进行设计的，具体在每个基因的内含子内选择一段序列作为探针，具体的探针由Advanced Cell Diagnostics公司设计。The designed probes are based on imprinted genes Z1 (Gnas), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z11 (Grb10), Z13 (Sgce) and Z16 (Snrpn/Snurf) For design, a sequence was selected as a probe within the intron of each gene. The specific probe was designed by Advanced Cell Diagnostics.
(4)将步骤(3)的探针与待测样本通过试剂盒进行RNA SCope原位杂交；(4) Use the probe in step (3) and the sample to be tested to perform RNA SCope in situ hybridization;
(5)信号扩增和苏木精染色，用显微镜成像分析印记基因的表达情况；(5) Signal amplification and hematoxylin staining, and microscopic imaging to analyze the expression of imprinted genes;
所述模型中的计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：The formula in the model for calculating the total expression of imprinted genes, the expression of missing imprinted genes and the abnormal expression of imprinted gene copy numbers is as follows:
总表达量＝(b+c+d)/(a+b+c+d)×100％；Total expression amount=(b+c+d)/(a+b+c+d)×100%;
正常印记基因表达量＝b/(b+c+d)×100％；Normal imprinted gene expression = b/(b+c+d)×100%;
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；Imprinted gene deletion gene expression level (LOI) = c/(b+c+d)×100%;
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；Gene expression amount with abnormal imprinted gene copy number (CNV) = d/(b+c+d)×100%;
其中，a、b、c、d如图1所示，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。Among them, a, b, c, and d are shown in Figure 1. The a is that after the cells are stained with hematoxylin, there is no marker in the nucleus and the imprinted gene is not expressed in the nucleus; the b is that after the cells are stained with hematoxylin, There is a red/brown mark in the nucleus, indicating that the imprinted gene is present in the nucleus; the c indicates that after hematoxylin staining of the cells, there are two red/brown markers in the nucleus, and the imprinted gene is missing; the d indicates that the cells were subjected to hematoxylin staining. After staining, there are more than two red/brown marks in the nucleus, and the nucleus has abnormal copy number of imprinted genes.
从图2(a)-图2(e)可以看出，从0级到IV级的样本中，印记缺失(细胞核内有两个信号点)和拷贝数异常(细胞核内有三个或以上信号点)的细胞比例随恶性程度的增加而逐渐增加。As can be seen from Figure 2(a)-Figure 2(e), in samples from level 0 to level IV, imprinting loss (two signal spots in the nucleus) and copy number abnormalities (three or more signal spots in the nucleus ) cells gradually increase with the degree of malignancy.
实施例2淋巴瘤患者淋巴结穿刺活检样本的印记基因分析Example 2 Imprinted gene analysis of lymph node biopsy samples from lymphoma patients
所述淋巴结穿刺活检样本是，通过穿刺取出可疑淋巴结组织，10％中性福尔马林溶液固定24h以上，其他检测方法同实施例1。The lymph node puncture biopsy sample is to remove suspicious lymph node tissue through puncture and fix it in 10% neutral formalin solution for more than 24 hours. Other detection methods are the same as in Example 1.
从图3(a)-图3(f)可以看出，Z1，Z4，Z5，Z6，Z8，Z11，Z13，Z16每个基因对恶性淋巴瘤的反应敏感性或者说对应于恶性淋巴瘤表达的印记缺失的强度和状态是不同的。As can be seen from Figure 3(a)-Figure 3(f), each gene Z1, Z4, Z5, Z6, Z8, Z11, Z13, Z16 is sensitive to malignant lymphoma or corresponds to the expression of malignant lymphoma. The intensity and status of the imprint loss are different.
具体每个印记基因对恶性淋巴瘤的敏感度如图4(a)-图4(h)，从图4(a)-图4(c)可以看出，印记基因Z1的印记缺失、拷贝数异常和表达量增加在恶性潜能阶段快速上升，在早期和中期恶性淋巴瘤阶段上升速度减缓，但是到晚期恶性淋巴瘤阶段又快速上升到很高水平；印记基因Z8的印记缺失和拷贝数异常在恶性潜能阶段开始出现，在早期到中期恶性淋巴瘤阶段逐渐上升，晚期恶性淋巴瘤中印记缺失继续增加，而拷贝数异常不再增加，印记基因Z8的表达量增加在恶性潜能阶段开始出现，在早期和中期恶性淋巴瘤阶段维持平稳，到晚期恶性淋巴瘤阶段又快速上升到较高水平；印记基因Z16的印记缺失、拷贝数异常和表达量增加在恶性潜能和早期恶性淋巴瘤阶段快速上升，在中期恶性淋巴瘤阶段维持平稳，到晚期恶性淋巴瘤阶段又快速上升到很高水平；The specific sensitivity of each imprinted gene to malignant lymphoma is shown in Figure 4(a)-Figure 4(h). From Figure 4(a)-Figure 4(c), it can be seen that the imprint loss and copy number of imprinted gene Z1 Abnormalities and increased expression increased rapidly in the malignant potential stage, slowed down in the early and intermediate malignant lymphoma stages, but rapidly rose to very high levels in the late malignant lymphoma stage; imprinting loss and copy number abnormalities of imprinted gene Z8 The malignant potential stage begins to appear, and gradually increases in the early to mid-stage malignant lymphoma stage. In late-stage malignant lymphoma, imprinting loss continues to increase, but the copy number abnormality no longer increases. The expression of imprinted gene Z8 begins to increase in the malignant potential stage. The early and intermediate malignant lymphoma stages remain stable, and then rapidly rise to a higher level in the late malignant lymphoma stage; imprinting loss, copy number abnormalities, and increased expression of the imprinted gene Z16 rapidly increase in the malignant potential and early malignant lymphoma stages. It remains stable at the intermediate malignant lymphoma stage, and rapidly rises to a very high level at the late malignant lymphoma stage;
从图4(d)-图4(h)可以看出，印记基因Z4的印记缺失在恶性潜能阶段开始少量出现，在早期和中期恶性淋巴瘤阶段缓慢上升，到晚期恶性淋巴瘤阶段快速上升，印记基因Z4的拷贝数异常和表达量增加在恶性潜能阶段开始出现，但是在恶性淋巴瘤的发展过程中没有明显上升；印记基因Z5的印记缺失、拷贝数异常和表达量增加在恶性潜能阶段开始出现，但是在恶性淋巴瘤的发展过程中没有明显上升；印记基因Z6的拷贝数异常在恶性潜能阶段开始少量出现，在早期和中期恶性淋巴瘤阶段缓慢上升，到晚期恶性淋巴瘤阶段快速上升，印记基因Z6的印记缺失和表达量增加在恶性潜能阶段开始出现，但是在恶性淋巴瘤的发展过程中没有明显上升；印记基因Z11的印记缺失、拷贝数异常和表达量增加在恶性潜能阶段开始少量出现，在早期和中期恶性淋巴瘤阶段缓慢上升，到晚期恶性淋巴瘤阶段上升速度加快；印记基因Z13的印记缺失在恶性潜能阶段开始出现，在早期和中期恶性淋巴瘤阶段缓慢上升，到晚期恶性淋巴瘤阶段上升速度加快，但是水平仍然很低，印记基因Z13的拷贝数异常在恶性潜能和早期恶性淋巴瘤阶段逐渐上升，在中晚期恶性淋巴瘤中没有明显上升，印记基因Z13的表达量增加在恶性潜能阶段开始出现，在早期和中期恶性淋巴瘤阶段缓慢上升，到晚期恶性淋巴瘤阶段上升速度加快，但水平仍然不高。As can be seen from Figure 4(d)-Figure 4(h), the imprinted deletion of imprinted gene Z4 begins to appear in small amounts in the malignant potential stage, slowly increases in the early and intermediate malignant lymphoma stages, and increases rapidly in the late malignant lymphoma stage. Abnormal copy number and increased expression of imprinted gene Z4 begin to appear in the malignant potential stage, but do not increase significantly during the development of malignant lymphoma; imprinting loss, abnormal copy number, and increased expression of imprinted gene Z5 begin in the malignant potential stage. appears, but does not increase significantly during the development of malignant lymphoma; abnormal copy number of imprinted gene Z6 begins to appear in small amounts in the malignant potential stage, increases slowly in the early and mid-stage malignant lymphoma stages, and increases rapidly in the late malignant lymphoma stage. The imprinting loss and expression increase of imprinted gene Z6 began to appear in the malignant potential stage, but did not increase significantly during the development of malignant lymphoma; the imprinting loss, copy number abnormality, and expression increase of imprinted gene Z11 began to appear in a small amount in the malignant potential stage. Appears, slowly rising in the early and middle stages of malignant lymphoma, and accelerating in the late stage of malignant lymphoma; imprinting loss of imprinted gene Z13 begins to appear in the malignant potential stage, rising slowly in the early and middle stages of malignant lymphoma, and rising in the late stage of malignant lymphoma. The rate of increase in lymphoma stages is accelerated, but the level is still very low. The abnormal copy number of imprinted gene Z13 gradually increases in the malignant potential and early malignant lymphoma stages, but does not increase significantly in intermediate and advanced malignant lymphomas. The expression of imprinted gene Z13 increases. It begins to appear in the malignant potential stage, rises slowly in the early and intermediate malignant lymphoma stages, and accelerates in the late malignant lymphoma stage, but the level is still not high.
实施例3 77例淋巴瘤样本的印记基因分析Example 3 Analysis of imprinted genes in 77 lymphoma samples
获取77例恶性淋巴瘤病人的组织包括淋巴结穿刺活检样本，检测方法同实施例1。Tissues including lymph node biopsy samples were obtained from 77 patients with malignant lymphoma, and the detection method was the same as in Example 1.
从图5(a)-图5(h)可以看出，77例淋巴瘤组织样本中8个探针的印记缺失和拷贝数异常的比例呈现从低到高的分布，根据不同探针的分布趋势，我们计算得到了图中虚线所示的分级标准，可以将每个探针的印记缺失、拷贝数异常和总表达量分别从低到高分成5个等级。It can be seen from Figure 5(a)-Figure 5(h) that the proportion of imprinting loss and copy number abnormality of 8 probes in 77 lymphoma tissue samples shows a distribution from low to high, according to the distribution of different probes Trend, we calculated the grading standard shown by the dotted line in the figure, which can divide the imprint loss, copy number abnormality and total expression of each probe into 5 grades from low to high respectively.
具体的分级如下：The specific classification is as follows:
从图5(a)可以看出，对于所述印记基因Z1，印记基因缺失表达量小于16％、印记基因拷贝数异常表达量小于1.5％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为16-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；As can be seen from Figure 5(a), for the imprinted gene Z1, the imprinted gene deletion expression amount is less than 16%, the imprinted gene copy number abnormal expression amount is less than 1.5%, or the total imprinted gene expression amount is less than 20%. Or at least two of them are grade 0, any one or at least two of the following: 16-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total expression of imprinted genes. The species is classified as level I, with any one or at least two of the following: 20-25% missing expression of imprinted genes, 2.5-5% abnormal expression of imprinted gene copy numbers, or 30-40% total imprinted gene expression. Grade III: any one or at least two of the following: 25-30% missing imprinted gene expression, 5-7% abnormal imprinted gene copy number expression, or 40-50% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 30%, abnormal imprinted gene copy number expression greater than 7%, or total imprinted gene expression greater than 50%, are grade IV;
从图5(b)可以看出，对于所述印记基因Z8，印记基因缺失表达量小于16％、印记基因拷贝数异常表达量小于1.5％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为16-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；As can be seen from Figure 5(b), for the imprinted gene Z8, the imprinted gene deletion expression amount is less than 16%, the imprinted gene copy number abnormal expression amount is less than 1.5%, or the total imprinted gene expression amount is less than 20%. Or at least two of them are grade 0, any one or at least two of the following: 16-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total expression of imprinted genes. The species is classified as level I, with any one or at least two of the following: 20-25% missing expression of imprinted genes, 2.5-5% abnormal expression of imprinted gene copy numbers, or 30-40% total imprinted gene expression. Grade III: any one or at least two of the following: 25-30% missing imprinted gene expression, 5-7% abnormal imprinted gene copy number expression, or 40-50% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 30%, abnormal imprinted gene copy number expression greater than 7%, or total imprinted gene expression greater than 50%, are grade IV;
从图5(c)可以看出，对于所述印记基因Z11，印记基因缺失表达量小于16％、印记基因拷贝数异常表达量小于1.5％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为16-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；As can be seen from Figure 5(c), for the imprinted gene Z11, the imprinted gene deletion expression amount is less than 16%, the imprinted gene copy number abnormal expression amount is less than 1.5%, or the total imprinted gene expression amount is less than 20%. Or at least two of them are grade 0, any one or at least two of the following: 16-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total expression of imprinted genes. The species is classified as level I, with any one or at least two of the following: 20-25% missing expression of imprinted genes, 2.5-5% abnormal expression of imprinted gene copy numbers, or 30-40% total imprinted gene expression. Grade III: any one or at least two of the following: 25-30% missing imprinted gene expression, 5-7% abnormal imprinted gene copy number expression, or 40-50% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 30%, abnormal imprinted gene copy number expression greater than 7%, or total imprinted gene expression greater than 50%, are grade IV;
从图5(d)可以看出，对于所述印记基因Z4，印记基因缺失表达量小于8％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为8-15％、印记基因拷贝数异常表达量为0.5-1.5％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-4％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于4％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；As can be seen from Figure 5(d), for the imprinted gene Z4, the imprinted gene deletion expression amount is less than 8%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%. Or at least two of them are grade 0, any one or at least two of the following: the expression of missing imprinted genes is 8-15%, the abnormal expression of imprinted genes is 0.5-1.5%, or the total expression of imprinted genes is 15-20% The species is grade I, any one or at least two of the following: 15-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total imprinted gene expression. Level III: any one or at least two of the following: 20-25% missing imprinted gene expression, 2.5-4% abnormal imprinted gene copy number expression, or 30-40% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 25%, abnormal imprinted gene copy number expression greater than 4%, or total imprinted gene expression greater than 40%, are grade IV;
从图5(e)可以看出，对于所述印记基因Z5，印记基因缺失表达量小于8％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为8-15％、印记基因拷贝数异常表达量为0.5-1.5％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-4％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于4％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；As can be seen from Figure 5(e), for the imprinted gene Z5, the imprinted gene deletion expression amount is less than 8%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%. Or at least two of them are grade 0, any one or at least two of the following: the expression of missing imprinted genes is 8-15%, the abnormal expression of imprinted genes is 0.5-1.5%, or the total expression of imprinted genes is 15-20% The species is grade I, any one or at least two of the following: 15-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total imprinted gene expression. Level III: any one or at least two of the following: 20-25% missing imprinted gene expression, 2.5-4% abnormal imprinted gene copy number expression, or 30-40% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 25%, abnormal imprinted gene copy number expression greater than 4%, or total imprinted gene expression greater than 40%, are grade IV;
从图5(f)可以看出，对于所述印记基因Z6，印记基因缺失表达量小于8％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为8-15％、印记基因拷贝数异常表达量为0.5-1.5％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-4％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于4％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；As can be seen from Figure 5(f), for the imprinted gene Z6, the imprinted gene deletion expression amount is less than 8%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%. Or at least two of them are grade 0, any one or at least two of the following: the expression of missing imprinted genes is 8-15%, the abnormal expression of imprinted genes is 0.5-1.5%, or the total expression of imprinted genes is 15-20% The species is grade I, any one or at least two of the following: 15-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total imprinted gene expression. Level III: any one or at least two of the following: 20-25% missing imprinted gene expression, 2.5-4% abnormal imprinted gene copy number expression, or 30-40% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 25%, abnormal imprinted gene copy number expression greater than 4%, or total imprinted gene expression greater than 40%, are grade IV;
从图5(g)可以看出，对于所述印记基因Z11，印记基因缺失表达量小于8％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为8-15％、印记基因拷贝数异常表达量为0.5-1.5％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-4％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于4％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；It can be seen from Figure 5(g) that for the imprinted gene Z11, the imprinted gene deletion expression amount is less than 8%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%. Or at least two of them are grade 0, any one or at least two of the following: the expression of missing imprinted genes is 8-15%, the abnormal expression of imprinted genes is 0.5-1.5%, or the total expression of imprinted genes is 15-20% The species is grade I, any one or at least two of the following: 15-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total imprinted gene expression. Level III: any one or at least two of the following: 20-25% missing imprinted gene expression, 2.5-4% abnormal imprinted gene copy number expression, or 30-40% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 25%, abnormal imprinted gene copy number expression greater than 4%, or total imprinted gene expression greater than 40%, are grade IV;
从图5(h)可以看出，对于所述印记基因Z13，印记基因缺失表达量小于8％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为8-15％、印记基因拷贝数异常表达量为0.5-1.5％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1.5-2.5％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2.5-4％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于4％或印记基因总表达量大于40％中的任意一种或至少两种为IV级。As can be seen from Figure 5(h), for the imprinted gene Z13, the imprinted gene deletion expression amount is less than 8%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%. Or at least two of them are grade 0, any one or at least two of the following: the expression of missing imprinted genes is 8-15%, the abnormal expression of imprinted genes is 0.5-1.5%, or the total expression of imprinted genes is 15-20% The species is grade I, any one or at least two of the following: 15-20% missing expression of imprinted genes, 1.5-2.5% abnormal expression of imprinted gene copy numbers, or 20-30% total imprinted gene expression. Level III: any one or at least two of the following: 20-25% missing imprinted gene expression, 2.5-4% abnormal imprinted gene copy number expression, or 30-40% total imprinted gene expression. Any one or at least two of the following: gene deletion and expression greater than 25%, abnormal imprinted gene copy number expression greater than 4%, or total imprinted gene expression greater than 40%, are grade IV.
从这77例淋巴瘤的样本综合分析可以得出：From the comprehensive analysis of these 77 lymphoma samples, it can be concluded that:
判断淋巴瘤的良恶性程度分为良性肿瘤、恶性潜能淋巴瘤、早期恶性淋巴瘤、中期恶性淋巴瘤和晚期恶性淋巴瘤；Judging the benign and malignant degree of lymphoma is divided into benign tumors, malignant potential lymphomas, early malignant lymphomas, intermediate malignant lymphomas and late malignant lymphomas;
判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；The result of judging the benign and malignant degree of lymphoma is that the imprinted gene deletion expression and imprinted gene copy number abnormal expression of imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 are less than level I or imprinted genes Z1, The imprinted gene deletion expression level of no more than 1 imprinted gene in Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I and the imprinted gene Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 If the abnormal expression of imprinted gene copy number of no more than 1 imprinted gene is grade I, it is a benign tumor;
判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则判断为恶性潜能淋巴瘤；The result of judging the benign and malignant degree of lymphoma is that the expression level of imprinted gene deletion of at least 2 imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level I, and the imprinted genes Z1, Z4, The abnormal expression amount of imprinted gene copy number in at least 2 imprinted genes of Z5, Z6, Z8, Z11, Z13 and Z16 is level I or does not exceed 1 in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16. The imprinted gene deletion expression level of imprinted genes is Level II and the abnormal expression level of imprinted gene copy number of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is Level II medium In any case, it is judged to be lymphoma with malignant potential;
判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期恶性淋巴瘤；The result for judging the benign and malignant degree of lymphoma is that the expression level of at least two imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is Level II, and the expression level of imprinted genes Z1, Z4, The abnormal expression amount of imprinted gene copy number is level II in at least 2 imprinted genes of Z5, Z6, Z8, Z11, Z13 and Z16 or does not exceed 1 in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 The imprinted gene deletion expression level of imprinted genes is Level III and the abnormal expression level of imprinted gene copy number of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is Level III medium In any case, it is early malignant lymphoma;
判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期恶性淋巴瘤；The result of judging the benign and malignant degree of lymphoma is that the expression level of at least two imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level III, and the expression level of imprinted genes Z1, Z4, The abnormal expression amount of imprinted gene copy number in at least 2 imprinted genes of Z5, Z6, Z8, Z11, Z13 and Z16 is level III or does not exceed 1 in imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 The imprinted gene deletion expression level of imprinted genes is level IV and the abnormal expression level of imprinted gene copy number of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level IV medium. In any case, it is mid-stage malignant lymphoma;
判断淋巴瘤的良恶性程度的结果为印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z4、Z5、Z6、Z8、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶性淋巴瘤。The result of judging the benign and malignant degree of lymphoma is that the imprinted gene deletion expression level of at least 2 imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is level IV or the imprinted genes Z1, Z4, Z5 If the abnormal expression of imprinted gene copy number of at least 2 imprinted genes among , Z6, Z8, Z11, Z13 and Z16 is level IV, it is an advanced malignant lymphoma.
实施例4乳腺癌前哨淋巴结的印记基因分析Example 4 Analysis of imprinted genes in sentinel lymph nodes of breast cancer
通过穿刺获取乳腺癌患者的前哨淋巴结样本，10％中性福尔马林溶液固定24h以上。根据印记基因Z1(Gnas)、Z2(Igf2)、Z3(Peg10)、Z4(Igf2r)、Z5(Mest)、Z6(Plagl1)、Z8(Dcn)、Z9(Dlk1)、Z10(Gatm)、Z11(Grb10)、Z12(Peg3)、Z13(Sgce)、Z14(Slc38a4)、Z15(Diras3)和Z16(Snrpn/Snurf)设计探针，具体在每个基因的内含子内选择一段序列作为探针，具体的探针由Advanced Cell Diagnostics公司设计，其他检测方法实施例1。Sentinel lymph node samples from breast cancer patients were obtained by puncture and fixed in 10% neutral formalin solution for more than 24 hours. According to the imprinted genes Z1 (Gnas), Z2 (Igf2), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z9 (Dlk1), Z10 (Gatm), Z11 ( Grb10), Z12 (Peg3), Z13 (Sgce), Z14 (Slc38a4), Z15 (Diras3) and Z16 (Snrpn/Snurf) were used to design probes. Specifically, a sequence was selected as a probe within the intron of each gene. The specific probe is designed by Advanced Cell Diagnostics Company. Example 1 of other detection methods.
从图6(a)-图6(c)可以看出，图6(a)为未转移的乳腺癌附近的淋巴结穿刺细胞，图6(b)为轻度转移的乳腺癌附近的淋巴结穿刺细胞，图6(c)为重度转移的乳腺癌附近的淋巴结穿刺细胞，在未转移的乳腺癌附近的淋巴结中只有个别印记缺失的细胞，没有发现拷贝数异常的癌细胞，在轻度转移的乳腺癌附近的淋巴结中存在少量印记基因拷贝数异常的癌细胞，在重度转移的乳腺癌附近的淋巴结中存在大量印记基因拷贝数异常的癌细胞。It can be seen from Figure 6(a)-Figure 6(c) that Figure 6(a) shows the lymph node puncture cells near non-metastatic breast cancer, and Figure 6(b) shows the lymph node puncture cells near mildly metastasized breast cancer. , Figure 6(c) shows lymph node puncture cells near severely metastasized breast cancer. In the lymph nodes near non-metastatic breast cancer, there are only cells with individual imprinting loss, and no cancer cells with abnormal copy number were found. In the lightly metastasized breast, There are a small number of cancer cells with abnormal imprinting gene copy numbers in the lymph nodes near the cancer, and there are a large number of cancer cells with abnormal imprinting gene copy numbers in the lymph nodes near severely metastasized breast cancer.
实施例5 42例癌症患者淋巴结穿刺的印记基因分析Example 5 Analysis of imprinted genes in lymph node biopsy of 42 cancer patients
获取乳腺癌、肺癌、胰腺癌患者的淋巴结穿刺样本，10％中性福尔马林溶液固定24h以上，其他检测方法同实施例1。Obtain lymph node puncture samples from patients with breast cancer, lung cancer, and pancreatic cancer, and fix them in 10% neutral formalin solution for more than 24 hours. Other detection methods are the same as in Example 1.
从图7(a)-图7(o)可以看出，42例癌症患者淋巴结穿刺样本中15个探针的印记缺失和拷贝数异常的比例呈现从低到高的分布，根据不同探针的分布趋势，我们计算得到了图中虚线所示的分级标准，可以将每个探针的拷贝数异常分别从低到高分成3个等级。It can be seen from Figure 7(a)-Figure 7(o) that the proportion of imprinting loss and copy number abnormality of 15 probes in the lymph node puncture samples of 42 cancer patients shows a distribution from low to high. According to the different probes, For the distribution trend, we calculated the grading standard shown by the dotted line in the figure, which can divide the copy number abnormalities of each probe into 3 grades from low to high.
从图7(a)可以看出，对于所述印记基因Z1，印记基因拷贝数异常表达量小于5％为0级，印记基因拷贝数异常表达量为5-10％为I级，印记基因拷贝数异常表达量大于10％为II级；As can be seen from Figure 7(a), for the imprinted gene Z1, the abnormal expression amount of imprinted gene copy number is less than 5%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 5-10%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 5-10%, which is grade I. The abnormal expression amount is greater than 10%, which is grade II;
从图7(b)可以看出，对于所述印记基因Z2，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(b), for the imprinted gene Z2, the abnormal expression amount of imprinted gene copy number is less than 2%, which is level 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is level I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is level I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(c)可以看出，对于所述印记基因Z3，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(c), for the imprinted gene Z3, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(d)可以看出，对于所述印记基因Z4，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；It can be seen from Figure 7(d) that for the imprinted gene Z4, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(e)可以看出，对于所述印记基因Z5，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(e), for the imprinted gene Z5, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(f)可以看出，对于所述印记基因Z6，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；It can be seen from Figure 7(f) that for the imprinted gene Z6, the abnormal expression amount of imprinted gene copy number is less than 2%, which is level 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is level I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(g)可以看出，对于所述印记基因Z8，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(g), for the imprinted gene Z8, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(h)可以看出，对于所述印记基因Z9，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；It can be seen from Figure 7(h) that for the imprinted gene Z9, the abnormal expression amount of imprinted gene copy number is less than 2%, which is level 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is level I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is level I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(i)可以看出，对于所述印记基因Z10，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(i), for the imprinted gene Z10, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(j)可以看出，对于所述印记基因Z11，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(j), for the imprinted gene Z11, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(k)可以看出，对于所述印记基因Z12，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(k), for the imprinted gene Z12, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(l)可以看出，对于所述印记基因Z13，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(l), for the imprinted gene Z13, if the abnormal expression amount of imprinted gene copy number is less than 2%, it is grade 0, and if the abnormal expression amount of imprinted gene copy number is 2-5%, it is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(m)可以看出，对于所述印记基因Z14，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；As can be seen from Figure 7(m), for the imprinted gene Z14, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(n)可以看出，对于所述印记基因Z15，印记基因拷贝数异常表达量小于2％为0级，印记基因拷贝数异常表达量为2-5％为I级，印记基因拷贝数异常表达量大于5％为II级；It can be seen from Figure 7(n) that for the imprinted gene Z15, the abnormal expression amount of imprinted gene copy number is less than 2%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 2-5%, which is grade I. The abnormal expression amount is greater than 5%, which is grade II;
从图7(o)可以看出，对于所述印记基因Z16，印记基因拷贝数异常表达量小于5％为0级，印记基因拷贝数异常表达量为5-10％为I级，印记基因拷贝数异常表达量大于10％为II级。As can be seen from Figure 7(o), for the imprinted gene Z16, the abnormal expression amount of imprinted gene copy number is less than 5%, which is grade 0, and the abnormal expression amount of imprinted gene copy number is 5-10%, which is grade I, and the abnormal expression amount of imprinted gene copy number is 5-10%, which is grade I. The abnormal expression amount is greater than 10%, which is grade II.
从这42例淋巴结穿刺样本综合分析可以得出：From the comprehensive analysis of these 42 lymph node biopsy samples, it can be concluded that:
判断淋巴结中癌细胞转移的程度分为未转移、轻度转移、重度转移；Determine the degree of cancer cell metastasis in lymph nodes into no metastasis, mild metastasis, and severe metastasis;
判断淋巴结中癌细胞转移的程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16的印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为未转移；The result of judging the degree of cancer cell metastasis in lymph nodes is the abnormal expression of imprinted gene copy number Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Abnormal imprinted gene copy number, all less than level I or no more than 1 imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 If the expression level is level I, it means it has not been transferred;
判断淋巴结中癌细胞转移的程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级，则为轻度转移；The result of judging the degree of cancer cell metastasis in the lymph node is the presence of at least 2 of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16. The abnormal expression level of imprinted gene copy number is level I or no more than 1 imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 If the abnormal expression level of imprinted gene copy number is level II, it is mild metastasis;
判断淋巴结中癌细胞转移的程度的结果为印记基因Z1、Z2、Z3、Z4、Z5、Z6、Z8、Z9、Z10、Z11、Z12、Z13、Z14、Z15和Z16中的至少2个印记基因的印记基因拷贝数异常表达量为II级，则为重度转移。The result of judging the degree of cancer cell metastasis in the lymph node is the presence of at least 2 of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16. The abnormal expression level of imprinted gene copy number is level II, which indicates severe metastasis.
综上所述，本发明所述检测模型和系统，以直观的方法表现了印记缺失在淋巴瘤和癌症淋巴转移病人的样本上的表现，通过对印记基因原位标记的方法，客观，直观，早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为淋巴瘤和癌症淋巴转移的诊断做出巨大贡献。In summary, the detection model and system of the present invention express the performance of imprinting loss in samples of patients with lymphoma and cancer lymphatic metastasis in an intuitive way. Through the method of in-situ labeling of imprinting genes, it is objective and intuitive. In the early stage, changes in imprinted (trace) genes can be accurately detected and quantitative models can be provided, making a huge contribution to the diagnosis of lymphoma and cancer lymphatic metastasis.
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The applicant declares that the present invention illustrates the detailed methods of the present invention through the above embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
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| CN201811270491.6ACN111105842B (en) | 2018-10-29 | 2018-10-29 | Grading model for detecting benign and malignant degrees of lymphoma and lymphatic metastatic carcinoma and application thereof |
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