发明领域field of invention
本发明属于基因工程及临床医学领域，涉及与常见型先天肠管无神经节细胞症发生相关的血浆微小核糖核酸（microRNA，miRNA）标志物及其应用。The invention belongs to the fields of genetic engineering and clinical medicine, and relates to plasma microRNA (miRNA) markers and applications thereof related to the occurrence of common congenital intestinal aganglionosis.
背景技术Background technique
常见型先天肠管无神经节细胞症（NCA）是小儿外科常见的消化道畸形，在先天性消化道畸形的发生中仅次于先天性肛门直肠畸形。CA以消化道远端神经节细胞缺如为主要特征，主要表现为肠神经系统发育过程中肠神经迁移障碍或迁移后发育异常，从而导致远端肠管神经节细胞缺如，远端肠管功能受限，最终以排便困难，腹胀及肠梗阻为主要临床症状。CA根据其狭窄段的长短主要分为短段型、常见型、长段型及全结肠型。其中，NCA是指肠管病变段位于肛门口开始至乙状结肠远端。基于NCA的高发病率，病情相对较轻，良好预后的基础，本发明以此型作为研究对象。目前我国统计NCA发病率约为1:3000，男女比大约为4:1，即每年有近一万的新生儿会出现该种出生缺陷。NCA的发病原因目前尚不完全明了，主要认为：在胚胎发育过程中，胚胎第5周神经母细胞开始沿迷走神经干由头侧向尾侧迁移，于胚胎第12周到达消化道远端，在此过程中任何原因导致神经母细胞迁移发生停顿即可造成远端肠管肠壁神经节细胞缺如。病理解剖提示直肠及远端结肠狭窄，近端肠管扩张，狭窄段肠壁黏膜下层及黏膜肌层神经节细胞缺如。狭窄段肠壁胆碱能受体及肾上腺能β受体含量较正常肠段减少，导致肠管及内括约肌痉挛，并且缺乏正常的肠蠕动，因此会形成功能性肠梗阻。长期慢性肠梗阻导致患儿食欲下降、营养吸收障碍、生长发育迟缓、贫血等，严重者引起小肠结肠炎、肠穿孔及多器官功能衰竭，危及患儿生命，给患儿及其家庭带来严重后果。Common congenital aganglionosis of the bowel (NCA) is a common digestive tract malformation in pediatric surgery, second only to congenital anorectal malformations in the occurrence of congenital digestive tract malformations. CA is mainly characterized by the absence of distal ganglion cells in the digestive tract, which is mainly manifested as enteric nerve migration disorder or abnormal development after migration during the development of the enteric nervous system, resulting in the absence of distal intestinal ganglion cells and impaired distal intestinal function. Finally, the main clinical symptoms are difficulty in defecation, abdominal distension and intestinal obstruction. According to the length of the stenosis, CA is mainly divided into short-segment type, common type, long-segment type and total colon type. Among them, NCA refers to the segment of intestinal lesion from the anus to the distal end of the sigmoid colon. Based on the high incidence of NCA, relatively mild condition and good prognosis, the present invention takes this type as the research object. At present, the incidence of NCA in my country is about 1:3000, and the ratio of male to female is about 4:1, that is, nearly 10,000 newborns will have this kind of birth defect every year. The pathogenesis of NCA is not yet fully understood. It is mainly believed that during embryonic development, neuroblasts begin to migrate from the cephalic to caudal side along the vagus nerve trunk at the 5th week of embryonic development, and reach the distal end of the digestive tract at the 12th week of embryonic development. Pause of neuroblast migration for any reason during the process can result in the absence of ganglion cells in the intestinal wall of the distal gut. Pathological anatomy revealed stenosis of the rectum and distal colon, dilatation of the proximal bowel, and absence of ganglion cells in the submucosa and muscularis mucosa of the stenosis. The content of cholinergic receptors and adrenergic β-receptors in the intestinal wall of the stenotic segment is lower than that of the normal segment, resulting in spasm of the intestinal tube and internal sphincter, and lack of normal intestinal peristalsis, thus forming functional intestinal obstruction. Long-term chronic intestinal obstruction leads to loss of appetite, nutrient absorption disorder, growth retardation, anemia, etc., and severe cases cause enterocolitis, intestinal perforation, and multiple organ failure, endangering the life of the child and bringing serious serious consequences to the child and his family. as a result of.
NCA患儿治疗多需手术，并且就目前来说手术是解决患儿临床症状的根治办法。但在临床工作中仍然会面临很多困难，特别是手术时机如何选择等。而新生儿常见型先天肠管无神经节细胞症的诊断相当困难，结合其临床表现：不排胎便及胎便排出延迟合并腹胀、梗阻、呕吐，直肠指检伴有气便排出，需考虑该疾病诊断，同时需对患儿进行一系列辅助检查如：X线，钡剂灌肠检查，肛门直肠测压，酶组织化学检查及活检等。以上辅助检查可以帮助诊断该疾病，但尚存在一些缺陷，如检查价格昂贵、射线辐射危险，操作繁琐且有一定的创伤性及风险。这都给患儿带来极大的痛苦并且给家庭带来严重的经济负担。更为重要的是，这类检查均基于不排胎便及胎便排出延迟等临床症状，而出现此类症状往往已在患病后数日常错过了最佳的早期手术时机，并给患儿带来了相当长时间的痛苦，寻找一种简单、准确、微创的早期常见型先天性肠管无神经节细胞症诊断方法意义重大。The treatment of children with NCA often requires surgery, and at present, surgery is the radical way to solve the clinical symptoms of children. However, there are still many difficulties in clinical work, especially how to choose the timing of surgery. However, the diagnosis of common congenital intestinal aganglionosis in neonates is quite difficult. Combined with its clinical manifestations: non-discharge of meconium and delayed discharge of meconium combined with abdominal distension, obstruction, vomiting, digital rectal examination accompanied by discharge of gas and feces, the diagnosis of this disease should be considered At the same time, a series of auxiliary examinations such as X-ray, barium enema, anorectal manometry, enzyme histochemical examination and biopsy should be carried out on the children. The above auxiliary examinations can help diagnose the disease, but there are still some defects, such as expensive examination, dangerous radiation, cumbersome operation and certain trauma and risk. All of these have brought great pain to the children and brought serious financial burdens to the families. More importantly, such examinations are based on clinical symptoms such as non-passage of meconium and delayed discharge of meconium, and the appearance of such symptoms often misses the best opportunity for early surgery a few days after the illness, and brings serious complications to the child. After suffering for quite a long time, it is of great significance to find a simple, accurate and minimally invasive early diagnosis method for common congenital intestinal aganglionosis.
微小核糖核酸（microRNA，即miRNA）是近年刚刚兴起的研究热点，它是一类长约19-23个核苷酸的单链RNA分子，多位于基因组非编码区，进化上高度保守，可在转录后水平对基因表达进行调节，并与动物的许多正常生理活动，如生物个体发育、组织分化、细胞凋亡以及能量代谢等密切相关，同时也与许多疾病的发生及发展存在着紧密的联系。自参与调控线虫时序发育的lin-4与let-7被发现以来，miRNA已逐渐成为调控mRNA稳定性和蛋白翻译的研究热点，分别在2002年和2003年两度入选Science杂志年度十大科技突破。现在预测miRNA至少能调控数千个人类基因，占所有基因的30%以上。随着研究的深入，越来越多的miRNA被发现。目前，miRNA与肿瘤的关系已成为研究的重点，已发现若干miRNA通过负调控基因的表达与慢性淋巴细胞性白血病、肺癌、乳腺癌、结肠癌高度相关。然而，血浆miRNA与常见型先天肠管无神经节细胞症等人类出生缺陷的相互关系尚未见报道。MicroRNA (miRNA) is a research hotspot that has just emerged in recent years. It is a type of single-stranded RNA molecule with a length of about 19-23 nucleotides. It is mostly located in the non-coding region of the genome and is highly conserved in evolution. The post-transcriptional level regulates gene expression and is closely related to many normal physiological activities of animals, such as individual development, tissue differentiation, cell apoptosis and energy metabolism, and is also closely related to the occurrence and development of many diseases . Since the discovery of lin-4 and let-7, which are involved in regulating the timing development of nematodes, miRNA has gradually become a research hotspot in regulating mRNA stability and protein translation, and was selected as one of the top ten scientific and technological breakthroughs of the Science magazine in 2002 and 2003, respectively. . It is now predicted that miRNA can regulate at least thousands of human genes, accounting for more than 30% of all genes. With the deepening of research, more and more miRNAs have been discovered. At present, the relationship between miRNAs and tumors has become the focus of research, and several miRNAs have been found to be highly correlated with chronic lymphocytic leukemia, lung cancer, breast cancer, and colon cancer through negative regulation of gene expression. However, the correlation between plasma miRNAs and human birth defects such as common congenital intestinal aganglionosis has not been reported.
最新的研究成果发现血浆中存在上百种的miRNA，性质稳定、含量丰富、易于定量检测，且存在显著的疾病特异性，在肺癌、结肠癌中已经证实血浆miRNA的表达谱可作为早期诊断的潜在生物标志物。这一发现令人振奋，血浆miRNA作为一类非编码调节性的小分子RNA有可能取代传统的特异蛋白为代表的生物标志物，开拓了生物标志物的新境界。该研究迅速引起国际媒体的广泛关注，路透社、合众社、《科学的美国人》、美国《技术评论》等都对该研究成果进行了专门报道，《Nature》杂志也在其网站首页的“最新研究进展”专栏中展示了这一最新研究进展。然而血浆中miRNA在常见型先天肠管无神经节细胞症早期诊断监测中的应用还未得到相应的关注，若能发现稳定的与常见型先天肠管无神经节细胞症发病相关的特异血浆miRNA作为生物标志物，并研发相应疾病的诊断、监测试剂盒，不仅能创造令人瞩目的经济效益，对我国出生缺陷的防治也将是一次强有力的推动。The latest research results have found that there are hundreds of miRNAs in plasma, which are stable in nature, abundant in content, easy to quantitatively detect, and have significant disease specificity. It has been confirmed in lung cancer and colon cancer that the expression profile of plasma miRNA can be used as an early diagnosis tool. potential biomarkers. This discovery is exciting. Plasma miRNA, as a type of non-coding regulatory small RNA, may replace the traditional biomarkers represented by specific proteins, opening up a new realm of biomarkers. The study quickly attracted widespread attention from the international media. Reuters, United Press, Scientific American, and Technology Review of the United States all made special reports on the research results. This latest research development is presented in the "Recent Research Development" column. However, the application of miRNA in plasma in the early diagnosis and monitoring of common congenital intestinal aganglionosis has not received corresponding attention. Markers, and the development of diagnostic and monitoring kits for corresponding diseases, will not only create remarkable economic benefits, but will also be a strong impetus to the prevention and treatment of birth defects in my country.
发明内容Contents of the invention
本发明的目的是为常见型先天肠管无神经节细胞症发生提供相关的血浆microRNA标志物。The purpose of the present invention is to provide relevant plasma microRNA markers for the occurrence of common type congenital intestinal tube aganglionosis.
本发明的另一个目的是为上述血浆microRNA标志物提供引物。Another object of the present invention is to provide primers for the above plasma microRNA markers.
本发明还有一个目的是为含有上述血浆提供microRNA标志物或其引物的应用。Another object of the present invention is to provide the application of microRNA markers or primers thereof for the plasma containing the above.
本发明再有一个目的是为含有上述血提供浆microRNA标志物或其引物的用于常见型肠管无神经节细胞症诊断或监测的试剂盒。Another object of the present invention is a kit for diagnosing or monitoring common intestinal tube aganglionosis containing the above-mentioned plasma microRNA markers or primers thereof.
本发明的目的是通过下列技术措施实现的：The purpose of the present invention is achieved through the following technical measures:
与常见型先天肠管无神经节细胞症相关的血浆microRNA标志物，选自hsa-miR-31、hsa-miR-147和hsa-miR-206中的多种。The plasma microRNA markers associated with the common type of congenital intestinal tube aganglionosis are selected from hsa-miR-31, hsa-miR-147 and hsa-miR-206.
所述的血浆microRNA标志物由hsa-miR-31、hsa-miR-147和hsa-miR-206构成。The plasma microRNA markers consist of hsa-miR-31, hsa-miR-147 and hsa-miR-206.
所述的血浆microRNA标志物，其中hsa-miR-31的序列为AGGCAAGAUGCUGGCAUAGCU（SEQIDNo.1），hsa-miR-147的序列为GUGUGUGGAAAUGCUUCUGC（SEQIDNo.2），hsa-miR-206的序列为UGGAAUGUAAGGAAGUGUGUGG（SEQIDNo.3）。The plasma microRNA marker, wherein the sequence of hsa-miR-31 is AGGCAAGAUGCUGGCAUAGCU (SEQ ID No.1), the sequence of hsa-miR-147 is GUGUGUGGAAAUGCUUCUGC (SEQ ID No.2), and the sequence of hsa-miR-206 is UGGAAUGUAAGGAAGUGUGUGG (SEQ ID No. .3).
所述的血浆microRNA标志物的引物，其中序列为SEQIDNo.1的标志物的上游引物为SEQIDNo.4，下游引物为SEQIDNo.5；序列为SEQIDNo.2的标志物的上游引物为SEQIDNo.6，下游引物为SEQIDNo.7；序列为SEQIDNo.3的标志物的上游引物为SEQIDNo.8，下游引物为SEQIDNo.9。The primers of the plasma microRNA markers, wherein the sequence is the upstream primer of the marker of SEQIDNo.1 is SEQIDNo.4, the downstream primer is SEQIDNo.5; the sequence is the upstream primer of the marker of SEQIDNo.2 is SEQIDNo.6, The downstream primer is SEQIDNo.7; the upstream primer of the marker whose sequence is SEQIDNo.3 is SEQIDNo.8, and the downstream primer is SEQIDNo.9.
所述的血浆microRNA标志物或其引物在制备常见型先天肠管无神经节细胞症诊断或监测试剂中的应用。所述试剂是能够测定这些血浆microRNA标志物在血浆中表达量的试剂。The application of the plasma microRNA marker or its primers in the preparation of diagnostic or monitoring reagents for common type congenital intestinal tube aganglionosis. The reagent is a reagent capable of measuring the expression levels of these plasma microRNA markers in plasma.
一种常见型肠管无神经节细胞症诊断或监测试剂盒，该试剂盒含有上述血浆microRNA标志物（hsa-miR-31、hsa-miR-147和hsa-miR-206中的多种）的引物。A diagnostic or monitoring kit for common intestinal aganglionosis, which contains primers for the above plasma microRNA markers (a variety of hsa-miR-31, hsa-miR-147 and hsa-miR-206) .
所述的诊断或监测试剂盒，该试剂盒含有上述引物（SEQIDNo.4和SEQIDNo.5，SEQIDNo.6和SEQIDNo.7，SEQIDNo.8和SEQIDNo.9）中的多对。The diagnostic or monitoring kit includes multiple pairs of the above primers (SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9).
所述的诊断或监测试剂盒，该试剂盒含有下列3对引物SEQIDNo.4和SEQIDNo.5，SEQIDNo.6和SEQIDNo.7，SEQIDNo.8和SEQIDNo.9。The diagnostic or monitoring kit includes the following 3 pairs of primers: SEQ ID No.4 and SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9.
试剂盒中除引物外的其他试剂可采用现有技术中相应检测技术的常用试剂。Common reagents of corresponding detection techniques in the prior art can be used for other reagents in the kit except the primers.
本发明详细描述如下：The present invention is described in detail as follows:
本发明人以标准操作程序（SOP）采集符合标准的血液样本，系统收集完整的人群基础信息和临床资料，并采用了RT-PCR方法、TaqmanmiRNAArray、Real-timePCR（Taqman探针和染料法）方法的一种或几种进行检测。The inventors used standard operating procedures (SOP) to collect standard blood samples, systematically collected complete population basic information and clinical data, and adopted RT-PCR method, TaqmanmiRNAArray, Real-timePCR (Taqman probe and dye method) methods One or more of them are detected.
具体来说研究的实验方法主要包括以下几个部分：Specifically, the experimental method of the research mainly includes the following parts:
一、研究对象选择和分组依据1. Research object selection and grouping basis
A组：健康对照组（n=100，20人芯片筛选，40人一期验证，40人独立人群验证）：Group A: healthy control group (n=100, 20 people for microarray screening, 40 people for first-stage verification, 40 people for independent crowd verification):
1.年龄在0至6月间；1. Age between 0 and 6 months;
2.无消化系统疾病；2. No digestive system disease;
3.无其它先天畸形；3. No other congenital malformations;
4.无其他全身性重大疾病。4. No other major systemic diseases.
B组：常见型先天肠管无神经节细胞症组（n=100，20人芯片筛选，40人一期验证，40人独立人群验证）：Group B: common congenital intestinal aganglionosis group (n=100, 20 people for microarray screening, 40 people for phase one verification, 40 people for independent population verification):
1.年龄在0至6月间；1. Age between 0 and 6 months;
2.经钡剂灌肠、结肠测压、术后病理证实病变段位于从肛门口至乙状结肠远端；2. Barium enema, colonic manometry, and postoperative pathology confirmed that the lesion was located from the anus to the distal end of the sigmoid colon;
3.排除短段型，长段型及全结肠型病变的患儿；3. Exclude children with short-segment type, long-segment type and total colon type lesions;
4.无其他伴随先天畸形；4. No other accompanying congenital deformities;
5.无其它消化系统疾病；5. No other digestive system diseases;
6.无其他全身性重大疾病。6. No other major systemic diseases.
二、血液血浆分离及前处理2. Blood plasma separation and pretreatment
（1）新鲜肝素抗凝血5ml于离心机3000rpm离心5min，取上清每100μl分装至洁净1.5mlEP管中。(1) Centrifuge 5ml of fresh heparin anticoagulated blood in a centrifuge at 3000rpm for 5min, and divide the supernatant into clean 1.5ml EP tubes by 100μl.
（2）向EP管中加入900μlTrizol，充分混匀后，12000转离心15min，立即取上清至一洁净1.5mlEP管中。(2) Add 900μl Trizol to the EP tube, mix thoroughly, centrifuge at 12000 rpm for 15min, and immediately take the supernatant into a clean 1.5ml EP tube.
（3）向EP管中加入1.5倍上清水相体积的无水乙醇，充分混匀后转移至离心柱，10000rpm离心15秒，弃下层废液。(3) Add 1.5 times the volume of supernatant water phase absolute ethanol to the EP tube, mix well, transfer to the spin column, centrifuge at 10000rpm for 15 seconds, and discard the waste liquid in the lower layer.
（4）在离心柱上加入700μlRWT缓冲液，10000rpm离心15秒，弃下层废液。(4) Add 700 μl of RWT buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the waste liquid in the lower layer.
（5）在离心柱上加入500μlRPE缓冲液，10000rpm离心15秒，弃下层液。重复一遍。(5) Add 500 μl RPE buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer. repeat.
（6）将离心柱加入一个新的2ml的管子，10000rpm离心1分钟，用于去除RPE缓冲液。(6) Put the spin column into a new 2ml tube and centrifuge at 10000rpm for 1 minute to remove the RPE buffer.
（7）将离心柱装在一个新的1.5ml的离心管中，并在柱子上加入50μlDEPC处理的水，离心1分钟。(7) Put the spin column in a new 1.5ml centrifuge tube, add 50μl DEPC-treated water to the column, and centrifuge for 1 minute.
（8）-70℃保存处理后的样本。(8) Store the processed samples at -70°C.
本发明实验中使用的离心柱和配套试剂（RWT缓冲液、RPE缓冲液）均来自QiagenmiRNeasyMiniKit（货号217004）这个试剂盒，下同。The spin column and supporting reagents (RWT buffer, RPE buffer) used in the experiment of the present invention are all from QiagenmiRNeasy MiniKit (product number 217004), the same below.
三、Real-timePCR方法测量血浆miRNAs表达量3. Real-time PCR method to measure the expression of plasma miRNAs
1.取经前处理的血浆，通过RNA逆转录反应得到cDNA样品。1. Take the pretreated plasma, and obtain cDNA samples through RNA reverse transcription reaction.
按下表所示配制反转录体系：Prepare the reverse transcription system as shown in the table below:
2.将PCR管反复颠倒混匀6次后做简短离心，冰上放置5分钟。2. Invert the PCR tube repeatedly for 6 times, then centrifuge briefly, and place it on ice for 5 minutes.
3.将PCR管放入PCR仪进行反转录，反应条件如下表所示：3. Put the PCR tube into the PCR machine for reverse transcription. The reaction conditions are shown in the table below:
反转录产物保存于4℃冰箱以用于下一步的预扩增。Reverse transcription products were stored in a 4°C refrigerator for pre-amplification in the next step.
4.逆转录之后的cDNA按下表反应体系配制进行预扩增：4. The cDNA after reverse transcription is prepared in the following reaction system for pre-amplification:
预扩增的反应条件如下表所示：The reaction conditions for pre-amplification are shown in the table below:
降至4℃后，将预扩增产物保存于4℃冰箱以用于下一步的Real-timePCR反应。After cooling down to 4°C, the pre-amplification products were stored in a refrigerator at 4°C for the next step of Real-timePCR reaction.
5.将预扩增产物简短离心后，加入0.1×TE(pH8.0)75μl，颠倒混匀后再做简短离心。预扩增产物可以直接用于下面的Real-timePCR。5. After briefly centrifuging the preamplified product, add 75 μl of 0.1×TE (pH 8.0), invert and mix well, and then perform brief centrifugation. The pre-amplified products can be directly used in the following Real-timePCR.
预扩增产物进行Real-timePCR按下表所示配制反应体系：Pre-amplified products were subjected to Real-timePCR to prepare the reaction system as shown in the following table:
考虑到吸液损失而放量12.5%。 Considering the loss of liquid absorption, the capacity is increased by 12.5%.
6.检测并比较健康对照组、常见型先天肠管无神经节细胞症组血浆样本中miRNAs表达量的差异。6. Detect and compare the differences in the expression of miRNAs in the plasma samples of the healthy control group and the common congenital intestinal aganglionosis group.
检测到的存在差异表达的健康对照和常见型先天肠管无神经节细胞症患者血浆miRNAs包括hsa-miR-31（SEQIDNo.1）、hsa-miR-147（SEQIDNo.2）、hsa-miR-206（SEQIDNo.3）。这些miRNAs在常见型先天肠管无神经节细胞症患者中的拷贝数都显著低于健康对照组，并且这些miRNAs在血浆中表达具有稳定性。The detected plasma miRNAs of healthy controls and patients with common congenital intestinal tube aganglionosis with differential expression include hsa-miR-31 (SEQ ID No.1), hsa-miR-147 (SEQ ID No.2), hsa-miR-206 (SEQ ID No. 3). The copy numbers of these miRNAs in patients with common congenital intestinal aganglionosis were significantly lower than those in healthy controls, and the expression of these miRNAs in plasma was stable.
四、Real-timePCR方法验证血浆miRNAs表达量4. Real-timePCR method to verify the expression of plasma miRNAs
1.设计3条目标miRNAs的引物：运用Stem-loopPCR方法设计引物。1. Design primers for 3 target miRNAs: use the Stem-loopPCR method to design primers.
2.加入荧光染料进行Real-timePCR反应。检测并比较非消化道畸形健康儿童、常见型先天肠管无神经节细胞症患儿血浆样本中miRNAs表达量的差异（病例和对照各100人）。2. Add fluorescent dyes for Real-time PCR reaction. To detect and compare the expression of miRNAs in the plasma samples of healthy children with non-digestive tract malformations and children with common congenital intestinal aganglionosis (100 cases and 100 controls).
3.选择独立人群（对照和病例各40人）进行Real-timePCR检测，结果一致的有3条miRNAs，具体为：hsa-miR-31、hsa-miR-147、hsa-miR-206。3. Select an independent population (40 persons for each control and case) for Real-time PCR detection, and there are 3 miRNAs with consistent results, specifically: hsa-miR-31, hsa-miR-147, hsa-miR-206.
因此，最终确认为存在差异表达的健康对照和常见型先天肠管无神经节细胞症患儿血浆miRNAs包括hsa-miR-31（SEQIDNo.1）、hsa-miR-147（SEQIDNo.2）和hsa-miR-206（SEQIDNo.3），具体引物见表1。其中，SEQIDNo.1、SEQIDNo.2和SEQIDNo.3在常见型先天肠管无神经节细胞症患儿血浆中的拷贝数都显著低于健康对照组，并且这些miRNAs在血浆中表达具有稳定性。采用SEQIDNo.1、SEQIDNo.2、SEQIDNo.3这3个构成的组合可以将对照组、常见型先天肠管无神经节细胞症组区分开。虽然这3条单独也可以分开两组人群，但如果只用1条，可能会有重合，而如果同时使用3条，即使1条差异不大，但另外2条表达差异都很大，就可以将其带入评分高的组。Therefore, the plasma miRNAs that were finally confirmed to be differentially expressed in healthy controls and children with common congenital intestinal agangliosis include hsa-miR-31 (SEQ ID No.1), hsa-miR-147 (SEQ ID No.2) and hsa-miR-31 (SEQ ID No.2) and hsa- miR-206 (SEQ ID No.3), see Table 1 for specific primers. Among them, the copy numbers of SEQIDNo.1, SEQIDNo.2 and SEQIDNo.3 in the plasma of children with common congenital intestinal aganglionosis were significantly lower than those in healthy controls, and the expression of these miRNAs in plasma was stable. The combination of SEQIDNo.1, SEQIDNo.2 and SEQIDNo.3 can be used to distinguish the control group and the common congenital intestinal aganglionosis group. Although these 3 items alone can separate the two groups of people, if only 1 item is used, there may be overlap, and if 3 items are used at the same time, even if the difference in 1 item is not large, but the expression of the other 2 items is very different, you can Bring it into the high scoring group.
五、诊断试剂盒制备方法5. Preparation method of diagnostic kit
根据上述一系列实验结果，本发明人还制备了一种能用于常见型先天肠管无神经节细胞症动态监测的诊断试剂盒，所述诊断试剂盒包含测定受试者血浆中稳定存在且可检测的成熟hsa-miR-31、hsa-miR-147、hsa-miR-206的引物和工具。诊断试剂盒包括一批血浆miRNAs引物，还可以包括Taq酶、三磷酸碱基脱氧核苷酸等试剂。According to the above series of experimental results, the inventors also prepared a diagnostic kit that can be used for dynamic monitoring of common type congenital intestinal aganglionosis. Primers and tools for detection of mature hsa-miR-31, hsa-miR-147, hsa-miR-206. The diagnostic kit includes a batch of plasma miRNAs primers, and may also include reagents such as Taq enzyme and base triphosphate deoxynucleotides.
本发明的有益效果：Beneficial effects of the present invention:
本发明人通过分离和比较正常对照和常见型先天肠管无神经节细胞症患儿血浆中的miRNAs，发现了血浆中存在可用于评估是否患有常见型先天肠管无神经节细胞症的特异性和敏感性（实施例6的ROC曲线提示具有较好的灵敏度，实施例7对其实际效果进行了验证，即常见型先天肠管无神经节细胞症患儿均被正确检测识别）的miRNA组合，因而提出了常见型先天肠管无神经节细胞症的血浆miRNA标志物组合，以及该血浆miRNA标志物或其引物在制备常见型先天肠管无神经节细胞症诊断或监测试剂中的应用，研制出可便于临床应用的常见型先天肠管无神经节细胞症诊断、监测试剂盒。By isolating and comparing miRNAs in the blood plasma of normal controls and children with common type congenital intestinal aganglionosis, the present inventors have found that there are specificity and Sensitivity (the ROC curve in Example 6 suggests that it has better sensitivity, and Example 7 has verified its actual effect, that is, children with common type congenital intestinal tube aganglionosis have been correctly detected and identified) miRNA combination, thus A combination of plasma miRNA markers for common types of congenital intestinal aganglionosis and the application of the plasma miRNA markers or their primers in the preparation of diagnostic or monitoring reagents for common types of congenital intestinal aganglionosis are proposed. Common type of congenital intestinal aganglionosis diagnosis and monitoring kits for clinical application.
本发明采用血浆miRNA作为常见型先天肠管无神经节细胞症评价的标志物的优越性在于：The present invention adopts the advantages of plasma miRNA as a marker for the evaluation of common type congenital intestinal tube aganglionosis in that:
（1）血浆miRNA是一种新型生物标志物，区别于传统生物标志物，不仅稳定、微创、易于检测，且定量精确，将大大提高常见型先天肠管无神经节细胞症诊断的敏感性和特异性，该类小分子RNA生物标志物的成功开发是对以蛋白为主的传统生物标志物的颠覆，将为出生缺陷的防治开创全新局面，为其他疾病生物标志物的研制提供借鉴。(1) Plasma miRNA is a new type of biomarker, which is different from traditional biomarkers. It is not only stable, minimally invasive, easy to detect, and accurate in quantification, but will greatly improve the sensitivity and Specificity, the successful development of this type of small molecule RNA biomarkers is a subversion of traditional protein-based biomarkers, which will create a new situation for the prevention and treatment of birth defects and provide reference for the development of other disease biomarkers.
（2）本发明提供的血浆miRNA标志物可用于常见型先天肠管无神经节细胞症诊断标志物，可避免侵入性诊断，并可在早期对常见型先天肠管无神经节细胞症进行辅助诊断，从而为临床医生进一步深入检查提供依据，为快速准确掌握患者的疾病状态和病情严重程度、及时采取更具个性化的防治方案提供支持，延缓和阻止疾病进展。(2) The plasma miRNA markers provided by the present invention can be used as diagnostic markers for common types of congenital intestinal aganglionosis, which can avoid invasive diagnosis, and can assist in the early diagnosis of common types of congenital intestinal aganglionosis, In this way, it can provide a basis for further in-depth inspections by clinicians, provide support for quickly and accurately grasping the patient's disease status and severity, and timely adopt more personalized prevention and treatment plans, so as to delay and prevent the progression of the disease.
（3）本发明采用符合常见型先天肠管无神经节细胞症和健康对照人群的样本进行验证，证明这几种标志物表达量存在显著性差异并具有稳定性，以说明该标志物具有特异性，可作为标志物使用。(3) The present invention is verified by samples conforming to the common type of congenital intestinal aganglionosis and healthy control groups, and it is proved that there are significant differences and stability in the expression of these markers, so as to illustrate the specificity of the markers , which can be used as markers.
（4）本发明采用严密、多阶段的验证和评价体系，初期通过预实验筛选多种血浆miRNAs，应用Real-timePCR等方法进行二次验证和独立人群验证，采用分层评分系统对诊断结果进行标化，并在另一组独立人群中对血浆miRNA标志物和诊断试剂盒进行盲法评价，保证了该血浆miRNA生物标志物和诊断试剂盒的可靠性。(4) The present invention adopts a rigorous and multi-stage verification and evaluation system. In the initial stage, a variety of plasma miRNAs are screened through preliminary experiments, and Real-timePCR and other methods are used for secondary verification and independent crowd verification, and a hierarchical scoring system is used to evaluate the diagnostic results. Standardization and blind evaluation of plasma miRNA markers and diagnostic kits in another independent group of people ensured the reliability of the plasma miRNA biomarkers and diagnostic kits.
附图说明Description of drawings
图1肛管直肠测压：该图显示常见型先天肠管无神经节细胞症病例直肠被动性扩张后内括约肌松弛反射缺如。Figure 1 Anorectal manometry: This figure shows the absence of the internal sphincter relaxation reflex after passive dilation of the rectum in a case of common congenital intestinal aganglionosis.
图2灌肠造影检查：该图显示常见型先天肠管无神经节细胞症影像学表现为结肠远端狭窄、痉挛，提示无神经节细胞病变肠段位于肛门开始至乙状结肠远端。Figure 2 Enema examination: This figure shows that the common type of congenital intestinal aganglionosis shows the imaging manifestations of stenosis and spasm in the distal colon, suggesting that the intestinal segment with aganglion cell lesion is located from the beginning of the anus to the distal end of the sigmoid colon.
图3以hsa-miR-31、hsa-miR-147、hsa-miR-206作为标志物对健康对照和常见型先天肠管无神经节细胞症组进行区分。Figure 3 uses hsa-miR-31, hsa-miR-147, and hsa-miR-206 as markers to distinguish between healthy controls and common congenital intestinal aganglionosis.
图4个体血浆miRNAs表达水平波动性分析。Fig. 4 Fluctuation analysis of individual plasma miRNAs expression levels.
图5正常对照组和常见型先天肠管无神经节细胞症组之间的ROC曲线。Fig. 5 ROC curve between the normal control group and the common type congenital intestinal aganglionosis group.
具体实施方式detailed description
以下通过实施例对本发明作进一步的阐述。The present invention is described further below by embodiment.
实施例1研究对象选择和分组依据Embodiment 1 Research Object Selection and Grouping Basis
本发明人于2009年7月到2011年9月间从南京医科大学附属儿童医院等医院搜集符合要求的常见型先天肠管无神经节细胞症患儿及正常非先天性肠管无神经节细胞症儿童血液和组织样品（临床诊断标准参考见图1，图2），通过对样品资料的整理，从中选择了符合要求的100例健康对照（平均年龄：89.45±9.1天）、100例肠管常见型先天肠管无神经节细胞症患者（平均年龄：92.3±7.01天）作为Real-timePCR检测miRNA表达的实验对象。具体的样品归类标准如下：From July 2009 to September 2011, the inventor collected children with common congenital intestinal aganglionosis and normal non-congenital intestinal aganglionosis from the Children's Hospital Affiliated to Nanjing Medical University and other hospitals that met the requirements. Blood and tissue samples (refer to Figure 1 and Figure 2 for the reference of clinical diagnostic standards), and 100 cases of healthy controls (average age: 89.45±9.1 days) and 100 cases of congenital Patients with intestinal aganglionosis (mean age: 92.3±7.01 days) were used as experimental subjects for Real-timePCR detection of miRNA expression. Specific sample classification criteria are as follows:
A组：健康对照组（n=100，20人芯片筛选，40人一期验证，40人独立人群验证）：Group A: healthy control group (n=100, 20 people for microarray screening, 40 people for first-stage verification, 40 people for independent crowd verification):
1.年龄在0至6月间；1. Age between 0 and 6 months;
2.无消化系统疾病；2. No digestive system disease;
3.无其它先天畸形；3. No other congenital malformations;
4.无其他全身性重大疾病。4. No other major systemic diseases.
B组：常见型先天肠管无神经节细胞症组（n=100，20人芯片筛选，40人一期验证，40人独立人群验证）：Group B: common congenital intestinal aganglionosis group (n=100, 20 people for microarray screening, 40 people for phase one verification, 40 people for independent population verification):
7.年龄在0至6月间；7. Age between 0 and 6 months;
8.经钡剂灌肠、结肠测压、术后病理证实病变段位于从肛门口至乙状结肠远端；8. Barium enema, colon manometry, and postoperative pathology confirmed that the lesion was located from the anus to the distal end of the sigmoid colon;
9.排除短段型，长段型及全结肠型病变的患儿；9. Exclude children with short-segment type, long-segment type and total colon type lesions;
10.无其他伴随先天畸形；10. No other accompanying congenital deformities;
11.无其它消化系统疾病；11. No other digestive system diseases;
12.无其他全身性重大疾病。12. No other major systemic diseases.
实施例2研究对象病理诊断分型Embodiment 2 research object pathological diagnosis type
对既往有胎便排出延迟，反复排便困难、腹胀、低位肠梗阻的患儿使用肛管直肠测压、影像学检查、直肠壁组织学检查诊断肠管无神经节细胞症并初步明确其类型。肛管直肠测压法主要表现为肛门内括约肌松弛反射缺如；肛管节律性收缩明显减少；直肠内括约肌部静止压高于正常。影像学检查表现为：1.在病变段与扩张段之间有一明显移行分隔区，呈现“锥体”状；2.病变段自肛门开始向上延展至乙状结肠远端；3.病变段神经支配异常故可见有不规则收缩；4.钡剂潴留，超过24-48小时可能仍未排出。直肠壁组织学检查主要观察黏膜下及肌间神经丛有无神经节细胞及细胞发育程度。正常的神经节细胞核大、染色深、居正中、核仁明显、周围胞浆嗜碱性，而病变肠段即狭窄、痉挛肠管缺乏神经节细胞，神经丛增生。以上临床检查方法均存在其局限性，其中直肠肛管测压诊断准确性在儿童组高达95%以上，新生儿组存在假阴性和假阳性结果，患儿接受检查时需口服镇静剂。放射学方法对患儿有辐射损伤，对新生儿也有假阴性和假阳性的情况。直肠壁活检方法较为精确，但属于创伤性诊断方法，需接受麻醉，对患儿有明显创伤性，风险较大，难以普遍开展。临床工作中更多的患儿确诊的方法是术后对手术切除标本进行病理学检查，术后组织标本狭窄段未见神经节细胞者可诊断肠管无神经节细胞症并明确其病变类型。Anorectal manometry, imaging examination, and rectal wall histological examination were used to diagnose intestinal aganglionosis and preliminarily clarify its type for children with delayed meconium discharge, repeated defecation difficulties, abdominal distension, and low intestinal obstruction in the past. Anorectal manometry mainly shows the absence of internal anal sphincter relaxation reflex; the rhythmic contraction of anal canal is significantly reduced; the resting pressure of internal rectal sphincter is higher than normal. Imaging examination showed that: 1. There was an obvious transition zone between the lesion segment and the dilation segment, showing a "cone" shape; 2. The lesion segment extended upward from the anus to the distal end of the sigmoid colon; 3. The innervation of the lesion segment was abnormal Therefore, it can be seen that there are irregular contractions; 4. The retention of barium agent may not be discharged for more than 24-48 hours. The histological examination of the rectal wall mainly observed whether there were ganglion cells and the degree of cell development in the submucosal and myenteric plexus. Normal ganglion cells have large nuclei, deep staining, centered, prominent nucleoli, and basophilic cytoplasm around them, while the diseased intestinal segment is narrow, spasmodic, lacking ganglion cells, and hyperplasia of nerve plexus. The above clinical examination methods all have their limitations. Among them, the diagnostic accuracy of rectal and anal canal manometry is as high as 95% in the children group, and there are false negative and false positive results in the neonatal group, and the children need oral sedatives during the examination. Radiological methods have radiation damage to children, and also have false negatives and false positives for newborns. The rectal wall biopsy method is relatively accurate, but it is an invasive diagnostic method that requires anesthesia. It is obviously invasive to children and has a high risk, so it is difficult to carry out widely. In clinical work, more children are diagnosed by pathological examination of surgical resection specimens after surgery. If ganglion cells are not found in the narrow section of postoperative tissue samples, intestinal aganglionosis can be diagnosed and the lesion type can be clarified.
实施例3TaqmanmiRNAarray筛选Embodiment 3TaqmanmiRNAarray screening
制备cDNA样品：a）取100μl血浆；b）加入900μlTrizol，振荡混匀，4℃，12000转离心15分钟，弃下层废液；c）加入上清1.5倍体积的无水乙醇震荡混匀，转至离心柱，12000转离心15秒，弃下层废液；d）在离心柱上加入700μlRWT缓冲液，10000rpm离心15秒，弃下层废液。e）在离心柱上加入500μlRPE缓冲液，10000rpm离心15秒，弃下层液。f）重复e。g）将离心柱加入一个新的2ml的管子，10000rpm离心1分钟，用于去除RPE缓冲液。h）在柱子上加入50μlDEPC处理水12000rpm离心收集RNA。i）然后通过RNA逆转录反应得到cDNA。逆转录的反应体系包括4μl5×AMV缓冲液、2μl10mMdNTP混合液（Takara公司）、0.5μlRNA酶抑制剂（Takara公司）、1μlAMV（Takara公司）以及1.5μl单一miRNA对应的反向引物。反应步骤为16℃孵育15分钟，42℃反应1小时，85℃孵育5分钟。（若针对不同miRNA则采用对应的miRNA反向引物按上述步骤进行）Preparation of cDNA samples: a) Take 100 μl plasma; b) Add 900 μl Trizol, shake and mix well, centrifuge at 12,000 rpm at 4°C for 15 minutes, discard the lower layer of waste liquid; c) Add 1.5 times the volume of supernatant absolute ethanol, shake and mix, transfer To the spin column, centrifuge at 12000 rpm for 15 seconds, discard the lower waste liquid; d) add 700 μl RWT buffer to the spin column, centrifuge at 10000 rpm for 15 seconds, discard the lower waste liquid. e) Add 500 μl RPE buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer. f) Repeat e. g) Add the spin column to a new 2ml tube and centrifuge at 10,000rpm for 1 minute to remove the RPE buffer. h) Add 50 μl of DEPC-treated water to the column and collect RNA by centrifugation at 12000 rpm. i) Then cDNA is obtained by RNA reverse transcription reaction. The reverse transcription reaction system included 4 μl 5×AMV buffer, 2 μl 10mMdNTP mixture (Takara), 0.5 μl RNase inhibitor (Takara), 1 μl AMV (Takara) and 1.5 μl reverse primer corresponding to a single miRNA. The reaction steps were incubation at 16°C for 15 minutes, reaction at 42°C for 1 hour, and incubation at 85°C for 5 minutes. (If targeting different miRNAs, use the corresponding miRNA reverse primer and follow the steps above)
逆转录之后的cDNA按下表反应体系配制进行预扩增：The cDNA after reverse transcription was pre-amplified according to the following reaction system:
将预扩增产物简短离心后，加入0.1×TE（pH8.0）75μl，颠倒混匀后再做简短离心。After brief centrifugation of the preamplified product, add 75 μl of 0.1×TE (pH8.0), invert and mix well, and then perform brief centrifugation.
预扩增产物可以直接用于下面的实时荧光定量PCR（qPCR）。The preamplified products can be directly used in the following real-time fluorescent quantitative PCR (qPCR).
预扩增产物进行qPCR按下表所示配制反应体系：Pre-amplified products were subjected to qPCR to prepare the reaction system as shown in the table below:
考虑到吸液损失而放量12.5%。预扩增产物稀释倍数为4倍。 Considering the loss of liquid absorption, the capacity is increased by 12.5%. The dilution factor of pre-amplification products was 4 times.
检测并比较健康对照、常见型先天肠管无神经节细胞症血浆样本中miRNAs表达谱的差异，筛选出有4倍差异以上的miRNAs。经生物信息学分析和动物实验结果，选定其中3条成为候选并进行进一步验证，具体为：hsa-miR-31、hsa-miR-147、hsa-miR-206。Detect and compare the differences in the expression profiles of miRNAs in plasma samples of healthy controls and common congenital intestinal aganglionosis, and screen out miRNAs with a difference of more than 4 times. After bioinformatics analysis and animal experiment results, three of them were selected as candidates for further verification, specifically: hsa-miR-31, hsa-miR-147, and hsa-miR-206.
实施例4Real-timePCR方法测量血浆miRNA表达量Embodiment 4Real-timePCR method measures plasma miRNA expression
设计引物（表1）分别对80例健康对照、80例常见型先天肠管无神经节细胞症患儿的血浆进行各miRNAs的定量Real-timePCR检测。The primers (Table 1) were designed for the quantitative Real-time PCR detection of each miRNAs in the plasma of 80 healthy controls and 80 children with common congenital intestinal aganglionosis.
（1）制备cDNA样品：a）取100μl血浆；b）加入900μlTrizol，振荡混匀，4℃，12000转离心15分钟，弃下层废液；c）加入上清1.5倍体积的无水乙醇震荡混匀，转至离心柱，12000转离心15秒，弃下层废液；d）在离心柱上加入700μlRWT缓冲液，10000rpm离心15秒，弃下层废液。e）在离心柱上加入500μlRPE缓冲液，10000rpm离心15秒，弃下层液。f）重复e。g）将离心柱加入一个新的2ml的管子，10000rpm离心1分钟，用于去除RPE缓冲液。h）在柱子上加入50μlDEPC处理水12000rpm离心收集RNA.i）然后通过RNA逆转录反应得到cDNA。逆转录的反应体系包括4μl5×AMV缓冲液、2μl10mMdNTP混合液（Takara公司）、0.5μlRNA酶抑制剂（Takara公司）、1μlAMV（Takara公司）以及1.5μl单一miRNA对应的反向引物。反应步骤为16℃孵育15分钟，42℃反应1小时，85℃孵育5分钟；(1) Preparation of cDNA samples: a) Take 100 μl of plasma; b) Add 900 μl of Trizol, shake and mix, centrifuge at 12,000 rpm at 4°C for 15 minutes, and discard the lower layer of waste liquid; c) Add 1.5 times the volume of supernatant absolute ethanol, shake and mix Mix well, transfer to the spin column, centrifuge at 12000 rpm for 15 seconds, discard the lower waste liquid; d) add 700 μl RWT buffer to the spin column, centrifuge at 10000 rpm for 15 seconds, discard the lower waste liquid. e) Add 500 μl RPE buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer. f) Repeat e. g) Add the spin column to a new 2ml tube and centrifuge at 10,000rpm for 1 minute to remove the RPE buffer. h) Add 50 μl of DEPC-treated water to the column and centrifuge at 12000 rpm to collect RNA. i) Then obtain cDNA through RNA reverse transcription reaction. The reverse transcription reaction system included 4 μl 5×AMV buffer, 2 μl 10mMdNTP mixture (Takara), 0.5 μl RNase inhibitor (Takara), 1 μl AMV (Takara) and 1.5 μl reverse primer corresponding to a single miRNA. The reaction steps are incubation at 16°C for 15 minutes, reaction at 42°C for 1 hour, and incubation at 85°C for 5 minutes;
（2）Real-timePCR：染料法：取1μlcDNA，将cDNA倍比稀释，加入0.3μlTaq酶（Takara公司），1μl20×EVAGREEN，0.25μl10μM上述单一miRNA对应的正向引物，0.25μl10μM通用反向引物（URP），1.2μl25mMMgCl2，1.6μl2.5mMdNTP混合液（Takara公司），2μl10×PCR缓冲液，12.4μl纯水，20μl体系进行荧光定量PCR。10μlTaqManuniversalPCRmasterMix，6.6μlH2O，20μl体系进行q-PCR。仪器使用的都是ABIPrism7900荧光定量PCR仪，PCR的反应条件都是：95℃、5分钟进行1个循环→95℃、15秒，60℃、1分钟进行40个循环。检测并比较健康对照、常见型先天肠管无神经节细胞症患儿血浆样本中miRNA表达量的变化，各组样品血浆miRNA的表达量比值可用方程2–△G表示，其中△G=CTgroup1–CTgroup2。为保证各次实验间的可比性，我们在每板上都设置了U6，以其表达量作为内参调整计算表达量。(2) Real-timePCR: Dye method: Take 1 μl cDNA, dilute the cDNA in a two-fold ratio, add 0.3 μl Taq enzyme (Takara company), 1 μl 20×EVAGREEN, 0.25 μl 10 μM forward primer corresponding to the above single miRNA, 0.25 μl 10 μM universal reverse primer ( URP), 1.2 μl 25mMMgCl2 , 1.6 μl 2.5mMdNTP mixture (Takara Company), 2 μl 10×PCR buffer, 12.4 μl pure water, 20 μl system for fluorescent quantitative PCR. 10 μl TaqManuniversalPCRmasterMix, 6.6 μl H2 O, 20 μl system for q-PCR. All the instruments used were ABIPrism7900 fluorescent quantitative PCR instruments, and the reaction conditions of PCR were: 95°C, 5 minutes for 1 cycle → 95°C, 15 seconds, 60°C, 1 minute for 40 cycles. Detect and compare the changes of miRNA expression levels in plasma samples of healthy controls and children with common congenital intestinal tube aganglionosis. The ratio of plasma miRNA expression levels of samples in each group can be expressed by Equation 2– △G , where △G=CTgroup1 – CTgroup2 . In order to ensure the comparability between experiments, we set U6 on each plate, and used its expression level as an internal reference to adjust and calculate the expression level.
从结果分析得出，hsa-miR-31、hsa-miR-147、hsa-miR-206这三条miRNA在各组间均有显著差别（图3A、图3B、图3C）。非参数的趋势性检验也显示了相同的差异。From the analysis of the results, it was found that the three miRNAs hsa-miR-31, hsa-miR-147, and hsa-miR-206 were significantly different among the groups (Fig. 3A, Fig. 3B, Fig. 3C). Nonparametric tests for trend also showed the same difference.
表1miRNAs的引物序列Table 1 Primer sequences of miRNAs
实施例5个体血浆miRNA表达量的稳定性分析Example 5 Stability analysis of individual plasma miRNA expression
采用实施例4的方法对八名儿童的血浆miRNA水平的稳定性进行评价。和实施例1同样的采集方法采集研究对象连续三次血浆（间隔时间为2周，间隔期内无疾病）。结果显示，血浆中hsa-miR-31、hsa-miR-147、hsa-miR-206这三个miRNA表达水平较稳定（图4）。这些都提示了个体血浆miRNA的表达量较为稳定，具备作为诊断/监测标志物的特性。The method of Example 4 was used to evaluate the stability of plasma miRNA levels in eight children. The same collection method as in Example 1 was used to collect the plasma of the research subjects for three consecutive times (the interval was 2 weeks, and there was no disease during the interval). The results showed that the expression levels of three miRNAs, hsa-miR-31, hsa-miR-147, and hsa-miR-206, were relatively stable in plasma (Figure 4). These all suggest that the expression level of individual plasma miRNA is relatively stable and has the characteristics of being a diagnostic/monitoring marker.
实施例6miRNA组合对常见型先天肠管无神经节细胞症的判断Example 6 Judgment of miRNA combination on common type congenital intestinal tube aganglionosis
根据上述Real-timePCR方法，本发明人通过对病例和对照组血浆样品的miRNAs表达水平的分析，以正常对照组miRNAs表达量的四分位数为阈值，对hsa-miR-31、hsa-miR-147、hsa-miR-206进行评分，进一步求得总得分，以此绘制ROC曲线来评估预测的灵敏性和特异性，进而评估这3个miRNAs低表达或高表达对常见型先天肠管无神经节细胞症的评估能力。ROC分析结果显示，hsa-miR-31、hsa-miR-147、hsa-miR-206以91.66%的AUC（ROC曲线下面积）将正常对照组和常见型先天肠管无神经节细胞症组分开；灵敏度：88.46%，特异度：78.26%（图5）。According to the above-mentioned Real-timePCR method, the present inventors analyzed the expression levels of miRNAs in plasma samples of cases and control groups, and took the quartile of miRNAs expression in the normal control group as the threshold value to hsa-miR-31, hsa-miR -147, hsa-miR-206 were scored, and the total score was further obtained, so as to draw the ROC curve to evaluate the sensitivity and specificity of prediction, and then evaluate the effect of low or high expression of these three miRNAs on the common type of congenital intestinal aneurosis Ability to assess gangliocytosis. ROC analysis results showed that hsa-miR-31, hsa-miR-147, and hsa-miR-206 separated the normal control group from the common congenital intestinal aganglionosis group with an AUC (area under the ROC curve) of 91.66%; Sensitivity: 88.46%, specificity: 78.26% (Figure 5).
在上述一系列研究结果的基础上，本发明人证明了采用hsa-miR-31、hsa-miR-147、hsa-miR-206能够很好地将对照和常见型先天肠管无神经节细胞症患儿分开。On the basis of the above series of research results, the inventors have proved that the use of hsa-miR-31, hsa-miR-147, and hsa-miR-206 can well separate the control and common congenital intestinal aganglion separate.
实施例7miRNA分层评分和独立人群盲法验证Example 7 miRNA hierarchical scoring and independent population blind verification
当对hsa-miR-31、hsa-miR-147、hsa-miR-206这三个标志物的表达水平进行分层评分时（四分位数分层后累加），能够对是否患有常见型先天肠管无神经节细胞症进行评估，表述为积分越低，确认为常见型先天肠管无神经节细胞症的风险越高。When the expression levels of the three markers hsa-miR-31, hsa-miR-147, and hsa-miR-206 are stratified and scored (accumulated after quartile stratification), it is possible to determine whether the common type Congenital intestinal aganglionosis was evaluated, expressed as the lower the score, the higher the risk of confirming the common type of congenital intestinal aganglionosis.
表3三个miRNAs四分位数得分并求和Table 3 Three miRNAs quartile scores and summation
注：每个miRNA按表达量的四分位数分为四个等级0、1、2、3，最低的0分，最高的3分，3条miRNA综合可以得分为0～9分，而常见型先天肠管无神经节细胞症组绝大多数得分很低（表达量低），而正常对照组绝大多数得分很低。举例来说，如有一个样本评分为9分（最高的分值组），则其不可能为常见型先天肠管无神经节细胞症病例，而应为正常对照。Note: Each miRNA is divided into four grades 0, 1, 2, and 3 according to the quartile of expression, the lowest score is 0, and the highest score is 3. The vast majority of patients with congenital intestinal aganglionosis had very low scores (low expression level), while the majority of normal controls had very low scores. For example, if there is a sample with a score of 9 (the highest score group), it cannot be a case of common congenital intestinal aganglionosis, but should be a normal control.
针对得出的评估分值（即根据具体得分判断其分组，得高分的归入对照组，得低分的归入常见型先天肠管无神经节细胞症组；比较而言，≥7算高分，≤2算低分），对另一组独立采集的人群进行盲法首诊，即采用双盲试验，对独立人群（另一医院采集的40例儿童）同时进行常规诊断分析和血浆样品的miRNA检测，结果显示通过3个miRNA检测评分，可以将常见型先天肠管无神经节细胞症及对照儿童很好的区分开（40例随机选择样本中有7例评分较低（≤2分），其中达到0分（最低分）的有3例，这3例经病理诊断均为常见型先天肠管无神经节细胞症，其余评分较高（≥7分）的均为非先天性肠管无神经节细胞症正常儿童），提示这几种miRNA可作为评估常见型先天肠管无神经节细胞症的标志物。According to the obtained evaluation score (that is, to judge the group according to the specific score, those with high scores are classified into the control group, and those with low scores are classified into the common congenital intestinal aganglionosis group; in comparison, ≥7 is considered high score, ≤2 is considered as low score), another group of independently collected people was blinded for the first visit, that is, a double-blind test was adopted, and routine diagnostic analysis and plasma samples were performed on an independent group (40 children collected from another hospital) at the same time. miRNA detection, the results showed that the common type of congenital intestinal tube aganglion cell disease and control children can be well distinguished by 3 miRNA detection scores (7 cases of 40 randomly selected samples had low scores (≤2 points) , of which 3 cases reached 0 points (the lowest score), and these 3 cases were pathologically diagnosed as common congenital intestinal aganglionosis, and the rest with higher scores (≥7 points) were all non-congenital intestinal aneurosis normal children with ganglionosis), suggesting that these miRNAs can be used as markers for the evaluation of common congenital intestinal aganglionosis.
实施例8用于常见型先天肠管无神经节细胞症诊断或监测试剂盒的制作Example 8 is used for the manufacture of common type congenital intestinal tube aganglionosis diagnosis or monitoring kit
该miRNA试剂盒的制作工艺和操作流程主要基于RT-PCR、Real-timePCR技术。The manufacturing process and operation process of the miRNA kit are mainly based on RT-PCR and Real-timePCR technologies.
首先通过测序的方法和Real-timePCR方法确定正常人和常见型先天肠管无神经节细胞症患儿血浆中有一个以上拷贝的miRNA。然后通过定量PCR等技术筛选与常见型先天肠管无神经节细胞症相关的一类血浆miRNA，作为预测是否患有常见型先天肠管无神经节细胞症以及诊断病变程度的指标。最后筛选出对应的血浆miRNA的数量控制在几条，这是在预实验的基础上做出的最优化的精简。此试剂盒包括一批血浆miRNA引物，其中miRNA的引物包括hsa-miR-31、hsa-miR-147、hsa-miR-206、U6的正反向引物（见表1）。还可以有相关PCR技术常用的试剂，如Taq酶、PCR缓冲液、MgCl2、三磷酸碱基脱氧核苷酸混合液、染料等试剂，这些试剂也可采用相应的市售产品。此试剂盒的价值在于只需要一次抽出少量（2ml）血液，即可检测血浆miRNA标志物的变化，可用于常见型先天肠管无神经节细胞症发生的辅助诊断，并易于进行动态监测和观察治疗效果。Firstly, by sequencing and Real-time PCR, it was determined that there were more than one copy of miRNA in the plasma of normal people and children with common congenital intestinal tube aganglionosis. Then, quantitative PCR and other techniques were used to screen a type of plasma miRNA associated with common congenital intestinal aganglionosis, as an indicator for predicting whether to suffer from common congenital intestinal aganglionosis and the degree of diagnosing the lesion. Finally, the number of corresponding plasma miRNAs was screened and controlled to a few, which was optimized and streamlined on the basis of the pre-experiment. This kit includes a batch of plasma miRNA primers, among which the miRNA primers include hsa-miR-31, hsa-miR-147, hsa-miR-206, U6 forward and reverse primers (see Table 1). Reagents commonly used in related PCR techniques can also be used, such as Taq enzyme, PCR buffer, MgCl2 , base triphosphate deoxynucleotide mixture, dyes and other reagents, and these reagents can also use corresponding commercially available products. The value of this kit is that it only needs to draw a small amount (2ml) of blood at a time to detect changes in plasma miRNA markers. It can be used for auxiliary diagnosis of common congenital intestinal tube aganglionosis, and it is easy to carry out dynamic monitoring and observation treatment. Effect.
具体试剂盒组成如下：The specific kit composition is as follows:
引物也可以是以下三对引物中的二对或三对：SEQIDNO.4和SEQIDNO.5，SEQIDNO.6和SEQIDNO.7，SEQIDNO.8和SEQIDNO.9，各10μM0.25μl。The primers can also be two or three pairs of the following three pairs of primers: SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, 10 μM 0.25 μl each.
试剂盒中还可以含有0.3μlTaq酶，1μl20×EVAGREEN，1.2μl25mMMgCl2，1.6μl2.5mMdNTP混合液，2μl10×PCR缓冲液，12.4μl纯水。The kit may also contain 0.3 μl Taq enzyme, 1 μl 20×EVAGREEN, 1.2 μl 25 mM MgCl2 , 1.6 μl 2.5 mM dNTP mixture, 2 μl 10×PCR buffer, and 12.4 μl pure water.
试剂盒中还可以含有内参U6的正反向引物一对（表1）。The kit can also contain a pair of forward and reverse primers for the internal reference U6 (Table 1).
或者试剂盒中除正向引物外还含有1μl10μM通用反向引物，10μlTaqMan通用PCR混合液，6.6μlH2O。Alternatively, in addition to the forward primer, the kit also contains 1 μl of 10 μM universal reverse primer, 10 μl of TaqMan universal PCR mixture, and 6.6 μl of H2 O.
试剂盒中的组分除引物外的试剂可以采用现有技术中用于miRNA含量检测的相应试剂。The reagents of the components in the kit except the primers can be the corresponding reagents used in the prior art for the detection of miRNA content.
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