CITED4和/或METRN在椎间盘退变程度的鉴别诊断中的应用Application of CITED4 and/or METRN in differential diagnosis of intervertebral disc degeneration相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请要求享有以下申请文件的优先权：2023年03月08日提交的申请号为2023102156761、名称为“CITED4和/或METRN在椎间盘退变程度的鉴别诊断中的应用”的发明专利申请，其内容以全文引用的方式并入本文。This application claims the priority of the following application documents: invention patent application with application number 2023102156761 filed on March 8, 2023 and entitled “Application of CITED4 and/or METRN in the differential diagnosis of the degree of intervertebral disc degeneration”, the contents of which are incorporated herein by reference in their entirety.
技术领域Technical Field本发明属于生物医药技术领域，具体地，本发明涉及CITED4和/或METRN在椎间盘退变程度的鉴别诊断中的应用。The present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to the application of CITED4 and/or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.
背景技术Background Art椎间盘退行性病变(Intervertebral disc degeneration，IDD)是由于年龄增长，运动系统功能减退，腰椎的稳定性降低，椎间盘所受应力增加，发生的退行性改变，又称为椎间盘退变，常见于中老年人。当椎间盘所承受的负荷超过了最大限度，纤维环破裂，最终可致髓核从中突出，引起腰腿痛。而椎间盘退行性病变是引起下腰背痛的主要原因之一。据统计，成年人中约有84％一生中至少会经历一次腰痛，其中由椎间盘退行性病变引起的腰痛占绝大多数。严重的椎间盘退行性病变甚至会引起下肢感觉障碍、大小便失禁等。椎间盘退行性病变已成为全球致残的重要因素，且随着世界人口老龄化，椎间盘退行性病变威胁健康的问题日益严重。因此，椎间盘退行性病变的早期诊断以及椎间盘退变的程度的量化对于及时干预治疗椎间盘退行性病变并延缓椎间盘退行性病变的进一步发展具有十分重要的临床价值。Intervertebral disc degeneration (IDD) is a degenerative change that occurs due to aging, decreased motor function, reduced stability of the lumbar spine, and increased stress on the intervertebral disc. It is also called disc degeneration and is common in middle-aged and elderly people. When the load on the intervertebral disc exceeds the maximum limit, the annulus fibrosus ruptures, and eventually the nucleus pulposus protrudes from it, causing low back pain and leg pain. Intervertebral disc degeneration is one of the main causes of low back pain. According to statistics, about 84% of adults will experience low back pain at least once in their lifetime, of which low back pain caused by intervertebral disc degeneration accounts for the vast majority. Severe intervertebral disc degeneration can even cause lower limb sensory impairment, incontinence, etc. Intervertebral disc degeneration has become an important factor in global disability, and with the aging of the world's population, the problem of intervertebral disc degeneration threatening health is becoming increasingly serious. Therefore, early diagnosis of disc degeneration and quantification of the degree of disc degeneration have very important clinical value for timely intervention and treatment of disc degeneration and delaying the further development of disc degeneration.
临床上对椎间盘退行性疾病的诊断目前仍然主要以影像学病变诊断为主，即采用核磁共振及X射线等影像学技术，通过观察椎间盘异常改变的情况判断椎间盘组织的退变程度。然而与经典的病理组织学诊断及基于病理组织学的分子病理诊断技术相比，影像学诊断虽然特异性较好且无创安全，但组织的影像学特征改变缓慢，将影像学特征作为椎间盘退行性病变的标志在探索治疗方案的前期基础研究及其后期临床诊疗方案的优化方面仍然存在一定的局限性。在更深层次的细胞和分子层面探索更多的椎间盘退行性病变诊断生物标志物，可能对其临床诊疗具有潜在的意义和价值。分子生物学的异常改变通常伴随甚至早于疾病组织形态学的变化，基因及蛋白水平的变化相较于疾病组织形态学通常也可以更加灵敏的反应。研究表明，髓核组织的退变早于其它椎间盘组织，与细胞老化、凋亡、自噬、细胞外基质稳态的改变、髓核中水分减少密切相关。而软骨细胞在成年人髓核中占有较大比例，承担了较大的负荷，是维持椎间盘生物学活性的重要成分。因此，探索退变髓核组织中软骨细胞的分子特征是该领域的热点。At present, the diagnosis of intervertebral disc degeneration is still mainly based on imaging lesions, that is, using imaging techniques such as nuclear magnetic resonance and X-ray to judge the degree of disc tissue degeneration by observing abnormal changes in the disc. However, compared with the classic pathological histological diagnosis and molecular pathological diagnosis techniques based on pathological histology, although imaging diagnosis has better specificity and is non-invasive and safe, the imaging characteristics of the tissue change slowly. Using imaging characteristics as a sign of intervertebral disc degeneration still has certain limitations in the early basic research of exploring treatment plans and the optimization of clinical diagnosis and treatment plans in the later stage. Exploring more diagnostic biomarkers for intervertebral disc degeneration at a deeper cellular and molecular level may have potential significance and value for its clinical diagnosis and treatment. Abnormal changes in molecular biology are usually accompanied by or even earlier than changes in disease tissue morphology, and changes in gene and protein levels are usually more sensitive than disease tissue morphology. Studies have shown that the degeneration of nucleus pulposus tissue is earlier than other intervertebral disc tissues, and is closely related to cell aging, apoptosis, autophagy, changes in extracellular matrix homeostasis, and reduced water content in the nucleus pulposus. Chondrocytes account for a large proportion of the nucleus pulposus in adults, bear a large load, and are an important component for maintaining the biological activity of the intervertebral disc. Therefore, exploring the molecular characteristics of chondrocytes in degenerated nucleus pulposus tissue is a hot topic in this field.
目前，尚未见有关CITED4和/或METRN在椎间盘退变程度的鉴别诊断中的应用的相关研究或报道。Currently, there are no studies or reports on the application of CITED4 and/or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.
发明内容Summary of the invention
为了克服目前本领域存在的上述技术问题，本发明提供了CITED4和/或METRN在椎间盘退变程度的鉴别诊断中的应用。In order to overcome the above technical problems existing in the current art, the present invention provides the application of CITED4 and/or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.
本发明利用单细胞转录组测序技术和生物信息学分析技术，通过分析本发明的发明人临床收集得到的人椎间盘髓核组织的单细胞基因表达谱，分离、表征、识别了一种能反映椎间盘退变过程的软骨细胞亚群，并通过进一步的研究确定了该细胞亚群所表达的新的特征基因CITED4、METRN，所述特征基因对椎间盘髓核组织退变的程度的鉴别诊断具有较高的诊断价值，对椎间盘髓核组织病理性退变的发生发展具有标记意义。The present invention utilizes single-cell transcriptome sequencing technology and bioinformatics analysis technology, and by analyzing the single-cell gene expression profile of human intervertebral disc nucleus pulposus tissue clinically collected by the inventor of the present invention, separates, characterizes, and identifies a chondrocyte subpopulation that can reflect the intervertebral disc degeneration process, and through further research, determines the new characteristic genes CITED4 and METRN expressed by the cell subpopulation. The characteristic genes have a high diagnostic value for the differential diagnosis of the degree of intervertebral disc nucleus pulposus tissue degeneration, and have a marking significance for the occurrence and development of pathological degeneration of intervertebral disc nucleus pulposus tissue.
为了实现上述目的，本发明采用了如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
本发明的第一方面提供了检测样本中CITED4和/或METRN表达水平的试剂在制备鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的产品中的应用。The first aspect of the present invention provides the use of a reagent for detecting the expression level of CITED4 and/or METRN in a sample in the preparation of a product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
进一步，所述试剂包括：Further, the reagent includes:
检测样本中CITED4和/或METRN的mRNA表达水平的试剂；Reagents for detecting the mRNA expression level of CITED4 and/or METRN in a sample;
检测样本中CITED4和/或METRN的蛋白表达水平的试剂；或A reagent for detecting the protein expression level of CITED4 and/or METRN in a sample; or
检测样本中CITED4和/或METRN阳性表达细胞数的试剂。A reagent for detecting the number of CITED4 and/or METRN positively expressed cells in a sample.
进一步，所述检测样本中CITED4和/或METRN的mRNA表达水平的试剂包括：Furthermore, the reagent for detecting the mRNA expression level of CITED4 and/or METRN in the sample includes:
特异性扩增CITED4和/或METRN的引物；或Primers that specifically amplify CITED4 and/or METRN; or
特异性识别CITED4和/或METRN的探针。Probes that specifically recognize CITED4 and/or METRN.
进一步，所述检测样本中CITED4和/或METRN的蛋白表达水平的试剂包括：Furthermore, the reagent for detecting the protein expression level of CITED4 and/or METRN in the sample includes:
特异性结合CITED4和/或METRN编码的蛋白的抗体；或An antibody that specifically binds to a protein encoded by CITED4 and/or METRN; or
特异性结合CITED4和/或METRN编码的蛋白的亲和性蛋白。Affinity proteins that specifically bind to proteins encoded by CITED4 and/or METRN.
进一步，所述检测样本中CITED4和/或METRN阳性表达细胞数的试剂包括通过免疫组化实验检测CITED4和/或METRN阳性表达细胞数的试剂。Furthermore, the reagent for detecting the number of CITED4 and/or METRN positively expressed cells in a sample includes a reagent for detecting the number of CITED4 and/or METRN positively expressed cells by immunohistochemistry.
在本发明中，所述基因CITED4、METRN的信息如下：基因CITED4(Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4)的Gene ID为163732，基因METRN(meteorin,glial cell differentiation regulator)的Gene ID为79006，所述基因的详细信息可在https://www.ncbi.nlm.nih.gov/gene/获得。In the present invention, the information of the genes CITED4 and METRN are as follows: the Gene ID of gene CITED4 (Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4) is 163732, and the Gene ID of gene METRN (meteorin, glial cell differentiation regulator) is 79006. The detailed information of the genes can be obtained at https://www.ncbi.nlm.nih.gov/gene/.
在本发明中，所述基因CITED4、METRN包括人CITED4基因、人METRN基因以及人CITED4基因、人METRN基因的任何功能等同物的多核苷酸。可以是天然的或是人工合成的，或者使用可以表达CITED4、METRN的DNA片段的载体转染细胞获得。所述载体包括病毒载体、真核表达载体。所述病毒载体可以是任何适当的载体，包括但不限于：逆转录病毒载体、腺病毒载体、腺病毒相关病毒载体、疱疹病毒(例如，单纯疱疹病毒、痘苗病毒及EB病毒)载体、甲病毒载体。所述真核表达载体可以是任何适当的表达载体，包括但不限于：pCMV-Myc表达载体、pcDNA3.0表达载体、pcDNA3.1表达载体、pEGFP表达载体、pEF Bos表达载体、pTet表达载体、pTRE表达载体、或者在公知表达载体的基础上经改造的载体，比如pBin438、pCAMBIA1301等。In the present invention, the genes CITED4 and METRN include polynucleotides of human CITED4 gene, human METRN gene and any functional equivalents of human CITED4 gene and human METRN gene. They can be natural or artificially synthesized, or obtained by transfecting cells with vectors that can express DNA fragments of CITED4 and METRN. The vectors include viral vectors and eukaryotic expression vectors. The viral vectors can be any appropriate vectors, including but not limited to retroviral vectors, adenoviral vectors, adenovirus-associated viral vectors, herpes virus (e.g., herpes simplex virus, vaccinia virus and Epstein-Barr virus) vectors, and alphavirus vectors. The eukaryotic expression vectors can be any appropriate expression vectors, including but not limited to pCMV-Myc expression vectors, pcDNA3.0 expression vectors, pcDNA3.1 expression vectors, pEGFP expression vectors, pEF Bos expression vectors, pTet expression vectors, pTRE expression vectors, or vectors modified on the basis of known expression vectors, such as pBin438, pCAMBIA1301, etc.
进一步，所述引物同扩增引物，是指包含5-100个核苷酸的核酸片段，优选地，所述引物或扩增引物包含能起始酶促反应(例如，酶促扩增反应)的15-30个核苷酸，在本发明的具体实施方式中，所述引物是指特异性扩增基因CITED4和/或METRN的引物。Furthermore, the primer and amplification primer refer to a nucleic acid fragment containing 5-100 nucleotides. Preferably, the primer or amplification primer contains 15-30 nucleotides that can initiate an enzymatic reaction (e.g., an enzymatic amplification reaction). In a specific embodiment of the present invention, the primer refers to a primer that specifically amplifies the genes CITED4 and/or METRN.
进一步，所述探针是指能与另一分子的特定序列或亚序列或其它部分结合的分子。在本发明的具体实施方式中，所述探针是指特异性识别CITED4和/或METRN的探针。除非另有指出，探针通常是指能通过互补碱基配对与另一多核苷酸(往往称为靶多核苷酸)结合的多核苷酸探针。根据杂交条件的严格性，探针能和与该探针缺乏完全序列互补性的靶多核苷酸结合。杂交方式包括但不限于：溶液相、固相、混合相或原位杂交测定法。本发明中的示例性探针包括基因特异性DNA寡核苷酸探针，例如固定于微阵列基底上的微阵列探针、定量核酸酶保护检验探针、与分子条形码连接的探针、以及固定于珠上的探针。Further, the probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. In a specific embodiment of the present invention, the probe refers to a probe that specifically recognizes CITED4 and/or METRN. Unless otherwise indicated, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays. Exemplary probes in the present invention include gene-specific DNA oligonucleotide probes, such as microarray probes fixed on a microarray substrate, quantitative nuclease protection test probes, probes connected to molecular barcodes, and probes fixed on beads.
杂交反应的严格性可以由本领域普通技术人员容易的确定，而且通常根据探针长度、洗涤温度和盐浓度凭经验计算。一般而言，较长的探针要求较高的温度以正确退火，而较短的探针需要较低的温度。杂交通常依赖于当互补链存在于低于其解链温度的环境中时变性DNA重新退火的能力。探针和可杂交序列之间的期望同源性程度越高，可使用的相对温度也越高。结果是，推断出较高相对温度将趋向于使反应条件更为严格，而较低温度也就较不严格。The stringency of hybridization reaction can be easily determined by those of ordinary skill in the art, and is usually calculated empirically based on probe length, washing temperature and salt concentration. Generally speaking, longer probes require higher temperatures to anneal correctly, while shorter probes require lower temperatures. Hybridization usually depends on the ability of denatured DNA to reanneal when complementary chains are present in an environment below their melting temperature. The higher the degree of expected homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is inferred that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are also less stringent.
进一步，所述特异性结合CITED4和/或METRN编码的蛋白的试剂包括但不限于：抗体、亲和性蛋白，还包括与CITED4和/或METRN编码的蛋白特异性结合的肽、适配体和/或化合物。Furthermore, the reagents that specifically bind to proteins encoded by CITED4 and/or METRN include, but are not limited to, antibodies, affinity proteins, and also include peptides, aptamers and/or compounds that specifically bind to proteins encoded by CITED4 and/or METRN.
进一步，所述抗体是本领域众所周知的，是指针对抗原位点的特异性免疫球蛋白。本发明中所述的抗体是指与本发明所述的CITED4和/或METRN编码的蛋白特异性结合的抗体，可以根据本领域中的常规方法来制造抗体。抗体的形式包括多克隆抗体或单克隆抗体、抗体片段(诸如Fab、Fab'、F(ab')2和Fv片段)、单链Fv(scFv)抗体、多特异性抗体(诸如双特异性抗体)、单特异性抗体、单价抗体、嵌合抗体、人源化抗体、人抗体、包含抗体的抗原结合位点的融合蛋白，以及包含抗原结合位点的任何其他修饰的免疫球蛋白分子，只要该抗体表现出所需的生物学结合活性即可。Further, the antibody is well known in the art and refers to a specific immunoglobulin for an antigenic site. The antibody described in the present invention refers to an antibody that specifically binds to the protein encoded by CITED4 and/or METRN described in the present invention, and the antibody can be manufactured according to conventional methods in the art. The form of the antibody includes polyclonal antibodies or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen binding site of the antibody, and any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.
进一步，所述肽具有与靶物质(本发明所述的CITED4和/或METRN编码的蛋白)高度结合的能力，并且在热处理或化学处理期间不会发生变性。而且，由于其尺寸小，可以通过将其附接到其它蛋白上而用作融合蛋白。具体而言，因为可以特异性地附接到高分子蛋白链上，其可以用作诊断试剂盒和药物递送物质。Further, the peptide has the ability to highly bind to the target substance (protein encoded by CITED4 and/or METRN of the present invention) and will not be denatured during heat treatment or chemical treatment. Moreover, due to its small size, it can be used as a fusion protein by attaching it to other proteins. Specifically, because it can be specifically attached to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.
进一步，所述适配体是指一种由特定类型的单链核酸(DNA、RNA或修饰的核酸)组成的多核苷酸，所述单链核酸自身具有稳定的三级结构，并且具有能够以高亲和力和特异性与靶分子(本发明所述的CITED4和/或METRN编码的蛋白)结合的特性。如上所述，由于适配体可以像抗体那样特异性结合抗原性物质，但比蛋白更稳定、具有更简单的结构，并且是由易于合成的多核苷酸组成，因此可以代替抗体来使用。Further, the aptamer refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid), which has a stable tertiary structure and has the property of being able to bind to the target molecule (the protein encoded by CITED4 and/or METRN described in the present invention) with high affinity and specificity. As mentioned above, since the aptamer can specifically bind to antigenic substances like an antibody, but is more stable than a protein, has a simpler structure, and is composed of a polynucleotide that is easy to synthesize, it can be used instead of an antibody.
进一步，所述通过免疫组化实验检测CITED4和/或METRN阳性表达细胞数的试剂包括但不限于任何通过免疫组化实验检测CITED4和/或METRN阳性表达细胞数所需的试剂，例如，固定剂、缓冲液、显色液、粘附剂、封固剂、酶消化液、蔗糖溶液。其中，所述固定剂包括但不限于：甲醛、戊二醛、多聚甲醛、乙醇、HneFIX、丙酮；所述缓冲液包括但不限于：PBS缓冲液、柠檬酸盐缓冲液、EDTA缓冲液、TBS缓冲液；所述显色液包括但不限于：DAB显色液、4-氯-1-萘酚(4-Cl-1-Naphthol)显色液、3-氨基-9-乙基卡唑(3-amino-9-ethylcarbozole,AEC)显色液、TMB显色液、NBT显色液；所述粘附剂包括但不限于：明胶、树脂胶、多聚赖氨酸、商品化粘附剂；所述封固剂包括但不限于：脱脂奶粉、BSA、血清以及Fab片段单链二抗；所述酶消化液包括但不限于：胰蛋白酶消化液、胃蛋白酶消化液。Furthermore, the reagents for detecting the number of CITED4 and/or METRN positively expressing cells by immunohistochemistry experiments include but are not limited to any reagents required for detecting the number of CITED4 and/or METRN positively expressing cells by immunohistochemistry experiments, for example, fixatives, buffers, color developing solutions, adhesives, mounting agents, enzyme digestion solutions, and sucrose solutions. Wherein, the fixing agent includes but is not limited to: formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, HneFIX, acetone; the buffer includes but is not limited to: PBS buffer, citrate buffer, EDTA buffer, TBS buffer; the color developing solution includes but is not limited to: DAB color developing solution, 4-chloro-1-naphthol (4-Cl-1-Naphthol) color developing solution, 3-amino-9-ethylcarbozole (3-amino-9-ethylcarbozole, AEC) color developing solution, TMB color developing solution, NBT color developing solution; the adhesive includes but is not limited to: gelatin, resin glue, polylysine, commercial adhesive; the sealing agent includes but is not limited to: skimmed milk powder, BSA, serum and Fab fragment single-chain secondary antibody; the enzyme digestion solution includes but is not limited to: trypsin digestion solution, pepsin digestion solution.
进一步，所述鉴别诊断椎间盘退变程度是指鉴别诊断受试者椎间盘退变的程度，在本发明的具体实施方案中，所述鉴别诊断椎间盘退变程度尤其是指CITED4和/或METRN对中度和重度退变组织、轻度和重度退变组织、轻中度和重度退变组织的鉴别诊断，且经实验验证发现CITED4和/或METRN在鉴别诊断上述不同退变程度的组织中的AUC值均大于0.9，灵敏度和特异性均较高，具有优异的诊断效能。Furthermore, the differential diagnosis of the degree of intervertebral disc degeneration refers to the differential diagnosis of the degree of intervertebral disc degeneration of the subject. In a specific embodiment of the present invention, the differential diagnosis of the degree of intervertebral disc degeneration especially refers to the differential diagnosis of moderate and severe degenerative tissues, mild and severe degenerative tissues, and mild-moderate and severe degenerative tissues by CITED4 and/or METRN, and experimental verification has found that the AUC values of CITED4 and/or METRN in the differential diagnosis of the above-mentioned tissues with different degrees of degeneration are all greater than 0.9, with high sensitivity and specificity, and have excellent diagnostic efficacy.
进一步，所述评估椎间盘退变治疗疗效是指评估受试者对某种治疗方法或治疗药物的疗效，在本发明的具体实施方案中，所述评估椎间盘退变治疗疗效尤其是指CITED4和/或METRN通过对椎间盘退变受试者的中度和重度退变组织、轻度和重度退变组织、轻中度和重度退变组织进行鉴别诊断，进一步评估得出受试者对某种治疗方法或治疗药物的疗效，具有较好的临床应用前景。Furthermore, the evaluation of the therapeutic efficacy of intervertebral disc degeneration refers to evaluating the efficacy of a subject's treatment of a certain treatment method or therapeutic drug. In a specific embodiment of the present invention, the evaluation of the therapeutic efficacy of intervertebral disc degeneration especially refers to CITED4 and/or METRN performing differential diagnosis on moderate and severe degenerative tissues, mild and severe degenerative tissues, and mild-moderate and severe degenerative tissues of subjects with intervertebral disc degeneration, and further evaluating the efficacy of the subject's treatment of a certain treatment method or therapeutic drug, which has good clinical application prospects.
进一步，所述表达水平是指本发明中所述CITED4和/或METRN的绝对量或相对量，可以通过本领域技术人员熟知的多种技术确定本发明中所述CITED4和/或METRN的表达水平，特别地，可以通过使用本领域技术人员熟知的免疫组化检测方法对本发明中所述的CITED4和/或METRN的绝对量或相对量进行检测。Further, the expression level refers to the absolute amount or relative amount of CITED4 and/or METRN described in the present invention. The expression level of CITED4 and/or METRN described in the present invention can be determined by a variety of techniques well known to those skilled in the art. In particular, the absolute amount or relative amount of CITED4 and/or METRN described in the present invention can be detected by using immunohistochemical detection methods well known to those skilled in the art.
进一步，所述样本是指获自或衍生自目标受试者的组合物，其包含有待例如基于物理、生物化学、化学和/或生理学特征表征和/或鉴定的细胞实体和/或其他分子实体。该样本可以获自受试者的血液和生物来源的其他流体样本及组织样本，如活检组织样本或从其衍生的组织培养物或细胞。组织样本的来源可以是实体组织，如来自新鲜、冷冻和/或保藏的器官或组织样本、活检组织或吸出物；血液或任意血液组分；体液；来自个体妊娠或发育的任何时间的细胞；或血浆。术语样本包括在其获得后以任何方式处理过的生物样本，如经试剂处理、稳定化、或针对某些成分(如蛋白质或多核苷酸)富集、或包埋在用于切片目的的半固体或固体基质中。Further, the sample refers to a composition obtained from or derived from a target subject, which contains cell entities and/or other molecular entities to be characterized and/or identified, for example, based on physical, biochemical, chemical and/or physiological characteristics. The sample can be obtained from the subject's blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples, biopsy tissues or aspirates; blood or any blood component; body fluids; cells from any time of individual pregnancy or development; or plasma. The term sample includes biological samples that have been processed in any way after they are obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes.
进一步，所述样本包括但不限于：组织、血液、血清、血浆、血液来源的细胞、淋巴液、滑膜液、脑脊髓液、胸膜液、腹腔液、膀胱冲洗液、分泌物(例如，乳腺分泌物)、口腔冲洗液、拭子(例如，口腔拭子)、触碰准备物、细针穿刺物、细胞提取物及其组合，在本发明的具体实施方案中，所述样本优选为受试者来源的组织样本，更优选为受试者来源的退变椎间盘组织样本。Furthermore, the sample includes, but is not limited to, tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder washing fluid, secretions (e.g., breast secretions), oral washing fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspirations, cell extracts, and combinations thereof. In a specific embodiment of the present invention, the sample is preferably a tissue sample derived from a subject, more preferably a degenerative intervertebral disc tissue sample derived from a subject.
进一步，所述鉴别诊断是指基于与个体相关的一个或多个的症状、数据或其他信息，来对个体的健康状态或状况的发现、判断、鉴别或认知。在本发明的具体实施方式中，所述椎间盘退变程度的鉴别诊断是指对不同椎间盘退变程度的个体进行区分，尤其是指对中度和重度椎间盘退变、轻度和重度椎间盘退变、轻中度和重度退变椎间盘退变的个体的区分。Furthermore, the differential diagnosis refers to the discovery, judgment, identification or recognition of the health status or condition of an individual based on one or more symptoms, data or other information related to the individual. In a specific embodiment of the present invention, the differential diagnosis of the degree of intervertebral disc degeneration refers to the differentiation of individuals with different degrees of intervertebral disc degeneration, especially the differentiation of individuals with moderate and severe intervertebral disc degeneration, mild and severe intervertebral disc degeneration, mild-moderate and severe degenerative intervertebral disc degeneration.
进一步，所述试剂是通过测序技术、核酸杂交技术、核酸扩增技术、蛋白免疫技术、免疫组化技术检测受试者待测样本中CITED4和/或METRN表达水平。Furthermore, the reagent is used to detect the expression level of CITED4 and/or METRN in the test sample of the subject through sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, and immunohistochemistry technology.
进一步，所述测序技术为核酸测序技术，包括链终止子(Sanger)测序技术和染料终止子测序技术，本领域的普通技术人员将认识到，由于RNA在细胞中不太稳定并且在实验中更易受到核酸酶攻击，因此，在测序前通常将RNA逆转录成DNA，此外，所述测序技术还包括下一代测序技术(即深度测序/高通量测序技术)。Furthermore, the sequencing technology is a nucleic acid sequencing technology, including chain terminator (Sanger) sequencing technology and dye terminator sequencing technology. Ordinary technicians in this field will recognize that since RNA is less stable in cells and is more susceptible to nuclease attack in experiments, RNA is usually reverse transcribed into DNA before sequencing. In addition, the sequencing technology also includes next-generation sequencing technology (i.e., deep sequencing/high-throughput sequencing technology).
进一步，所述核酸杂交技术包括但不限于：原位杂交(ISH)、微阵列和Southern或Northern印迹。Furthermore, the nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blotting.
进一步，所述核酸扩增技术包括但不限于：聚合酶链式反应(PCR)、逆转录聚合酶链式反应(RT-PCR)、转录介导的扩增(TMA)、连接酶链式反应(LCR)、链置换扩增(SDA)、基于核酸序列的扩增(NASBA)。Furthermore, the nucleic acid amplification technology includes but is not limited to: polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).
进一步，所述蛋白免疫技术包括夹心免疫测定，例如夹心ELISA，其中使用识别CITED4和/或METRN上不同表位的两种抗体进行该标志物的检测；放射免疫测定(RIA)、直接、间接或对比酶联免疫吸附测定(ELISA)、酶免疫测定(EIA)、荧光免疫测定(FIA)、蛋白质印迹法、免疫沉淀法和基于任何颗粒的免疫测定(如使用金颗粒、银颗粒或乳胶颗粒、磁性颗粒或量子点)。Further, the protein immunoassay technique includes sandwich immunoassay, such as sandwich ELISA, in which two antibodies recognizing different epitopes on CITED4 and/or METRN are used to detect the marker; radioimmunoassay (RIA), direct, indirect or comparative enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), Western blotting, immunoprecipitation, and any particle-based immunoassay (eg, using gold, silver or latex particles, magnetic particles or quantum dots).
本发明的第二方面提供了一种用于鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的产品。A second aspect of the present invention provides a product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
进一步，所述产品包括检测样本中CITED4和/或METRN表达水平的试剂。Furthermore, the product includes a reagent for detecting the expression level of CITED4 and/or METRN in a sample.
进一步，所述产品包括检测试剂盒、生物芯片。Furthermore, the products include detection kits and biochips.
进一步，所述检测试剂盒包括特异性结合CITED4和/或METRN的引物、探针；Further, the detection kit includes primers and probes that specifically bind to CITED4 and/or METRN;
所述生物芯片包括固相载体、附着在固相载体上的特异性识别CITED4和/或METRN的探针。The biochip comprises a solid phase carrier and a probe attached to the solid phase carrier and capable of specifically recognizing CITED4 and/or METRN.
进一步，所述检测试剂盒还包括选自以下组的一种或多种物质：容器、使用说明书、阳性对照物、阴性对照物、缓冲剂、助剂或溶剂。Furthermore, the detection kit further comprises one or more substances selected from the following group: a container, instructions for use, a positive control substance, a negative control substance, a buffer, an auxiliary agent or a solvent.
进一步，所述检测试剂盒包括：RT-PCR检测试剂盒、ELISA检测试剂盒、蛋白芯片检测试剂盒、快速检测试剂盒、DNA芯片检测试剂盒、免疫组化检测试剂盒、或MRM(多反应监测)检测试剂盒。Furthermore, the detection kit includes: an RT-PCR detection kit, an ELISA detection kit, a protein chip detection kit, a rapid detection kit, a DNA chip detection kit, an immunohistochemistry detection kit, or an MRM (multiple reaction monitoring) detection kit.
进一步，所述检测试剂盒可进一步包含反转录聚合酶链式反应所必需的元件。RT-PCR检测试剂盒包含一对特异性针对编码标记物蛋白的基因的引物。各引物是具有特异性针对所述基因的核酸序列的核苷酸，其长度可为约7至50bp，更特别为约10-39bp。此外，所述试剂盒可进一步包含特异性针对对照基因的核酸序列的引物；优选地，所述RT-PCR检测试剂盒还可包含测试管或合适的器皿、反应缓冲液(不同pH值和镁浓度)、脱氧核苷酸(dNTP)、酶(例如Taq聚合酶和反转录酶)、脱氧核糖核酸酶抑制剂、核糖核酸酶抑制剂、DEPC-水、和无菌水。Further, the detection kit may further include the elements necessary for reverse transcription polymerase chain reaction. The RT-PCR detection kit includes a pair of primers specific for the gene encoding the marker protein. Each primer is a nucleotide with a nucleic acid sequence specific for the gene, and its length may be about 7 to 50bp, more particularly about 10-39bp. In addition, the kit may further include primers specific for the nucleic acid sequence of the control gene; preferably, the RT-PCR detection kit may also include a test tube or a suitable vessel, a reaction buffer (different pH values and magnesium concentrations), a deoxynucleotide (dNTP), an enzyme (such as Taq polymerase and reverse transcriptase), a deoxyribonuclease inhibitor, a ribonuclease inhibitor, DEPC-water, and sterile water.
进一步，所述检测试剂盒可包含用于操作DNA芯片所必需的元件。所述DNA芯片试剂盒可包含与基因或cDNA或相当于其片段的寡核苷酸结合的底物、及用于构建荧光标记的探针的试剂、药剂和酶。此外，所述底物可包含对照基因或cDNA或相当于其片段的寡核苷酸。Further, the detection kit may include elements necessary for operating a DNA chip. The DNA chip kit may include a substrate bound to a gene or cDNA or an oligonucleotide equivalent to a fragment thereof, and reagents, agents and enzymes for constructing a fluorescently labeled probe. In addition, the substrate may include a control gene or cDNA or an oligonucleotide equivalent to a fragment thereof.
在一些实施方案中，本发明公开的检测试剂盒可包含用于进行ELISA所必需的元件。所述ELISA检测试剂盒可包含特异性针对蛋白(本发明所述的CITED4和/或METRN编码的蛋白)的抗体。所述抗体具有针对标记物蛋白的高选择性和亲合力，与其他蛋白无交叉反应性，并且可以是单克隆抗体、多克隆抗体或重组抗体。此外，所述ELISA检测试剂盒可包含特异性针对对照蛋白的抗体。此外，所述ELISA检测试剂盒可进一步包含能够检测被结合的抗体的试剂，例如，标记的第二抗体、发色团、酶(例如，与抗体缀合)、及其底物或能够结合所述抗体的物质。In some embodiments, the detection kit disclosed in the present invention may include elements necessary for performing ELISA. The ELISA detection kit may include antibodies specific for proteins (proteins encoded by CITED4 and/or METRN described in the present invention). The antibody has high selectivity and affinity for marker proteins, no cross-reactivity with other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. In addition, the ELISA detection kit may include antibodies specific for control proteins. In addition, the ELISA detection kit may further include reagents capable of detecting the bound antibody, for example, a labeled second antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), and a substrate thereof or a substance capable of binding the antibody.
进一步，所述生物芯片也称为阵列，指包含连接的核酸或肽探针的固体支持物。阵列通常包含按照不同的已知位置连接至基底表面的多种不同的核酸或肽探针。这些阵列，也称为微阵列，通常可以利用机械合成方法或光引导合成方法来产生这些阵列，所述光引导合成方法合并了光刻方法和固相合成方法的组合。阵列可以包含平坦的表面，或者可以是珠子、凝胶、聚合物表面、诸如光纤的纤维、玻璃或任何其它合适的基底上的核酸或肽。可以以一定的方式来包装阵列，从而允许进行全功能装置的诊断或其它方式的操纵。Further, the biochip is also referred to as an array, which refers to a solid support comprising connected nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes connected to the substrate surface according to different known positions. These arrays, also referred to as microarrays, can typically be produced using mechanical synthesis methods or light-guided synthesis methods, which incorporate a combination of photolithography and solid phase synthesis methods. The array can comprise a flat surface, or can be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass or any other suitable substrate. The array can be packaged in a certain manner to allow for diagnosis of a fully functional device or manipulation of other methods.
微阵列是杂交阵列原件有序排列在基质上，所述杂交阵列原件诸如聚核苷酸探针(例如寡核苷酸)或结合剂(例如抗体)。所述基质可以是固体基质，例如，玻璃或二氧化硅玻片、珠、纤维光学粘结剂或半固态基质，例如硝酸纤维素膜。核苷酸序列可以是DNA、RNA或其中的任何排列。Microarrays are hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), arranged in an orderly manner on a substrate. The substrate can be a solid substrate, for example, a glass or silica slide, a bead, a fiber optic binder, or a semi-solid substrate, such as a nitrocellulose membrane. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.
在本发明中，所述生物芯片包括基因芯片、蛋白芯片；所述基因芯片包括固相载体；以及有序固定在所述固相载体上的寡核苷酸探针，所述的寡核苷酸探针特异性地对应于CITED4和/或METRN所示的部分或全部序列。所述蛋白芯片包括固相载体，以及固定在固相载体上的CITED4和/或METRN编码的蛋白的特异性抗体或配体。In the present invention, the biochip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier; and oligonucleotide probes fixed in order on the solid phase carrier, and the oligonucleotide probes specifically correspond to part or all of the sequences shown by CITED4 and/or METRN. The protein chip includes a solid phase carrier, and specific antibodies or ligands of proteins encoded by CITED4 and/or METRN fixed on the solid phase carrier.
进一步，所述抗体明确包括嵌合抗体(免疫球蛋白)，其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而重链和/或轻链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性。Further, the antibodies specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.
进一步，所述配体可包含能够特异性结合CITED4和/或METRN的肽、抗体或其片段、或适配体或寡核苷酸。本发明中使用的针对CITED4和/或METRN编码的蛋白的抗体以最广义使用，具体涵盖例如单克隆抗体、多克隆抗体、具有多表位特异性的抗体，多特异性抗体和抗体片段。此类抗体可以是嵌合的，人源化的，人的和合成的。只要所述片段能够保留与CITED4和/或METRN编码的蛋白的结合能力即可。Further, the ligand may comprise a peptide, an antibody or a fragment thereof, or an aptamer or an oligonucleotide that can specifically bind to CITED4 and/or METRN. The antibodies used in the present invention against proteins encoded by CITED4 and/or METRN are used in the broadest sense, specifically covering, for example, monoclonal antibodies, polyclonal antibodies, antibodies with multi-epitope specificity, multispecific antibodies and antibody fragments. Such antibodies may be chimeric, humanized, human and synthetic. As long as the fragment can retain The ability to bind to the protein encoded by CITED4 and/or METRN is sufficient.
本发明的第三方面提供了检测样本中CITED4和/或METRN表达水平的试剂在制备鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的系统和/或装置中的应用。The third aspect of the present invention provides the use of a reagent for detecting the expression level of CITED4 and/or METRN in a sample in the preparation of a system and/or device for differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic efficacy of intervertebral disc degeneration.
本发明的第四方面提供了一种鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的系统和/或装置。A fourth aspect of the present invention provides a system and/or device for differentially diagnosing the degree of intervertebral disc degeneration or evaluating the efficacy of treatment for intervertebral disc degeneration.
进一步，所述系统/装置包括处理器、输入模块、输出模块；Further, the system/device includes a processor, an input module, and an output module;
其中，处理器用于对输入的信息采用生物信息学方法进行逻辑运算；输入模块用于输入受试者样本中CITED4和/或METRN的表达水平，包含指令的计算机可读介质，所述指令在由所述处理器执行时在CITED4和/或METRN的输入表达水平上执行算法；输出模块用于输出受试者椎间盘退变的程度或椎间盘退变的治疗疗效。Among them, the processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the expression level of CITED4 and/or METRN in the subject's sample, and contains a computer-readable medium of instructions, which, when executed by the processor, executes an algorithm on the input expression level of CITED4 and/or METRN; the output module is used to output the degree of intervertebral disc degeneration of the subject or the therapeutic efficacy of intervertebral disc degeneration.
进一步，本发明中所述的受试者意指任何动物，还指人类和非人类的动物。术语非人类的动物包括所有脊椎动物，例如，哺乳动物，如非人灵长类动物(特别是高等灵长类动物)、绵羊、狗、啮齿类动物(如小鼠或大鼠)、豚鼠、山羊、猪、猫、兔、牛、和任何家畜或宠物；以及非哺乳动物，如鸡，两栖类，爬行动物等，在本发明的具体实施方式中，所述受试者优选为人。Further, the subject described in the present invention means any animal, and also refers to humans and non-human animals. The term non-human animals includes all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject is preferably a human.
本发明还提供了一种计算机可读存储介质。所述计算机可读存储介质上存储有计算机程序，所述计算机程序被处理器执行时实现本发明第四方面所述的系统和/或装置。The present invention further provides a computer-readable storage medium having a computer program stored thereon, and when the computer program is executed by a processor, the system and/or device according to the fourth aspect of the present invention is implemented.
进一步，所述系统/装置是用于区分不同级别的不同组件、元件、部件、部分或装配的一种方法。然而，如果其他词语可实现相同的目的，则可通过其他表达来替换所述词语。本领域所属技术领域的技术人员熟知，本发明可以实现为设备、方法或计算机程序产品。因此，本发明公开的内容可以具体实现为以下形式，即可以是完全的硬件、也可以是完全的软件(包括固件、驻留软件、微代码等)，还可以是硬件和软件结合的形式。此外，在一些具体实施例中，本发明还可以实现为在一个或多个计算机可读介质中的计算机程序产品的形式，该计算机可读介质中包含计算机可读的程序代码。Further, the system/device is a method for distinguishing different components, elements, parts, parts or assemblies at different levels. However, if other words can achieve the same purpose, the words can be replaced by other expressions. It is well known to those skilled in the art that the present invention can be implemented as an apparatus, method or computer program product. Therefore, the content disclosed in the present invention can be specifically implemented in the following forms, that is, it can be complete hardware, it can be complete software (including firmware, resident software, microcode, etc.), and it can also be a combination of hardware and software. In addition, in some specific embodiments, the present invention can also be implemented in the form of a computer program product in one or more computer-readable media, and the computer-readable medium contains computer-readable program code.
可以采用一个或多个计算机可读的介质的任意组合。计算机可读介质可以是计算机可读信号介质或者计算机可读存储介质。计算机可读存储介质例如可以是但不限于电、磁、光、电磁、红外线、或半导体的系统、装置或器件，或者任意以上的组合。在本发明中，所述计算机可读存储介质可以是任何包含或存储程序的有形介质，该程序可以被指令执行系统、装置或者器件使用或者与其结合使用。Any combination of one or more computer-readable media may be used. The computer-readable medium may be a computer-readable signal medium or a computer-readable storage medium. The computer-readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, device or device, or any combination thereof. In the present invention, the computer-readable storage medium may be any tangible medium containing or storing a program that may be used by or in conjunction with an instruction execution system, device or device.
本发明的第五方面提供了用于鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的生物标志物。A fifth aspect of the present invention provides a biomarker for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
进一步，所述生物标志物为CITED4和/或METRN。Furthermore, the biomarkers are CITED4 and/or METRN.
本发明的第六方面提供了生物标志物CITED4和/或METRN在鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效中的应用。The sixth aspect of the present invention provides the use of the biomarkers CITED4 and/or METRN in differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
在本发明中，只要采用本发明所述的生物标志物CITED4和/或METRN以鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效，这样的方法均落入本发明的保护范围内，并不局限于使用所述生物标志物CITED4和/或METRN的具体方法，只要能够实现或基本实现鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的目的，其均属于本发明的保护范围。In the present invention, as long as the biomarkers CITED4 and/or METRN described in the present invention are used to differentially diagnose the degree of intervertebral disc degeneration or evaluate the therapeutic efficacy of intervertebral disc degeneration, such methods fall within the protection scope of the present invention and are not limited to the specific methods of using the biomarkers CITED4 and/or METRN. As long as the purpose of differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic efficacy of intervertebral disc degeneration can be achieved or basically achieved, it falls within the protection scope of the present invention.
本发明的第七方面提供了一种鉴别诊断椎间盘退变程度或评估椎间盘退变治疗疗效的方法。A seventh aspect of the present invention provides a method for differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic efficacy of intervertebral disc degeneration.
进一步，所述方法包括如下步骤：Further, the method comprises the following steps:
(1)收集受试者来源的样本；(1) Collect samples from subjects;
(2)检测受试者来源的样本中CITED4和/或METRN的表达水平；(2) detecting the expression levels of CITED4 and/or METRN in samples derived from subjects;
(3)根据检测得到的CITED4和/或METRN的表达水平鉴别诊断受试者椎间盘退变程度或评估受试者椎间盘退变治疗疗效。(3) Differentiate and diagnose the degree of intervertebral disc degeneration in subjects or evaluate the therapeutic efficacy of intervertebral disc degeneration in subjects based on the expression levels of CITED4 and/or METRN detected.
本发明的第八方面提供了生物标志物CITED4和/或METRN在治疗和/或预防椎间盘退变中的应用。The eighth aspect of the present invention provides the use of the biomarkers CITED4 and/or METRN in the treatment and/or prevention of intervertebral disc degeneration.
在本发明中，只要采用本发明所述的生物标志物CITED4和/或METRN以治疗和/或预防椎间盘退变，这样的方法均落入本发明的保护范围内，并不局限于使用所述生物标志物CITED4和/或METRN的具体方法，只要采用CITED4和/或METRN能够实现或基本实现治疗和/或预防椎间盘退变的目的，其均属于本发明的保护范围。In the present invention, as long as the biomarkers CITED4 and/or METRN described in the present invention are used to treat and/or prevent intervertebral disc degeneration, such methods fall within the protection scope of the present invention and are not limited to the specific methods of using the biomarkers CITED4 and/or METRN. As long as the use of CITED4 and/or METRN can achieve or basically achieve the purpose of treating and/or preventing intervertebral disc degeneration, it falls within the protection scope of the present invention.
相对于现有技术，本发明具有的优点和有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
(1)本发明首次发现CITED4、METRN在不同退变程度的椎间盘退变组织中存在显著差异表达，其与椎间盘退变的程度具有极高的关联度，在本发明收集的临床样本中表现出对椎间盘退变程度的较高的鉴别诊断效能，准确性、灵敏度和特异性均较高，其中，CITED4分别用于鉴别诊断中度与重度、轻度与重度、轻中度与重度退变的AUC值分别为0.917、0.961、0.938，METRN分别用于鉴别诊断中度与重度、轻度与重度、轻中度与重度退变的AUC值为0.911、0.929、0.92，CITED4和METRN共同用于鉴别诊断中度与重度、轻度与重度、轻中度与重度退变的AUC值为0.933、0.953、0.949。表明CITED4、METRN能够作为鉴别诊断椎间盘退变程度的生物标志物，有效用于椎间盘退变程度的鉴别诊断中，进而在早期进行及时有效的干预和治疗。(1) The present invention first discovered that CITED4 and METRN were significantly differentially expressed in degenerative disc tissues of different degrees of degeneration, and that they were highly correlated with the degree of disc degeneration. In the clinical samples collected by the present invention, they showed a high differential diagnostic efficacy for the degree of disc degeneration, with high accuracy, sensitivity and specificity. The AUC values of CITED4 for differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs. severe degeneration were 0.917, 0.961 and 0.938, respectively; the AUC values of METRN for differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs. severe degeneration were 0.911, 0.929 and 0.92, respectively; the AUC values of CITED4 and METRN for differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs. severe degeneration were 0.933, 0.953 and 0.949, respectively. These results indicate that CITED4 and METRN can be used as biomarkers for differential diagnosis of the degree of intervertebral disc degeneration, and can be effectively used in the differential diagnosis of the degree of intervertebral disc degeneration, thereby allowing for timely and effective intervention and treatment in the early stages.
(2)本发明提供的CITED4和/或METRN对椎间盘退变程度的鉴别诊断的诊断效能较高，具有良好的开发为诊断方法的前景。此外，本发明为解决本领域存在的在临床实践中影像学诊断依赖于组织的影像学特征、而组织的影像学特征改变缓慢导致诊断结果滞后这一技术问题，以及疾病组织形态学的变化往往晚于分子生物学的异常改变导致的基于组织形态学的相关诊断方法的灵敏度较低这一技术问题提供了全新的解决策略。本发明从分子水平鉴定出的特征基因能够为椎间盘退变生物标志物的发掘、推广应用提供一定的理论依据，对椎间盘退行性病变的鉴别诊断、相关干预措施的效用评价以及了解该疾病的发病机制和病程进展均具有重要的应用意义。(2) The CITED4 and/or METRN provided by the present invention have high diagnostic efficacy for the differential diagnosis of the degree of intervertebral disc degeneration and have good prospects for development as diagnostic methods. In addition, the present invention provides a new solution strategy to solve the technical problems in the field that imaging diagnosis in clinical practice depends on the imaging characteristics of tissues, and the imaging characteristics of tissues change slowly, resulting in delayed diagnostic results, and the changes in disease tissue morphology are often later than the abnormal changes in molecular biology, resulting in low sensitivity of related diagnostic methods based on tissue morphology. The characteristic genes identified at the molecular level by the present invention can provide a certain theoretical basis for the discovery and promotion of biomarkers of intervertebral disc degeneration, and have important application significance for the differential diagnosis of intervertebral disc degeneration, the effectiveness evaluation of related intervention measures, and the understanding of the pathogenesis and progression of the disease.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS图1为人椎间盘髓核组织单细胞悬液制备质控结果图；FIG1 is a diagram showing the quality control results of the preparation of a single cell suspension of human intervertebral disc nucleus pulposus tissue;
图2为人椎间盘髓核组织单细胞亚群结果图；FIG2 is a diagram showing the results of single cell subpopulations in human intervertebral disc nucleus pulposus tissue;
图3为展示人椎间盘髓核组织软骨细胞第8亚群标记分子CITED4和METRN的mRNA表达情况的小提琴图；FIG3 is a violin plot showing the mRNA expression of CITED4 and METRN, marker molecules of the 8th subpopulation of chondrocytes in the nucleus pulposus of human intervertebral disc;
图4为人椎间盘髓核组织中Aggrecan(ACAN)表达阳性的细胞被鉴定为软骨细胞的结果图，其中，左图：100X，右图：400X；FIG4 is a diagram showing the result of Aggrecan (ACAN)-positive cells in human intervertebral disc nucleus pulposus tissue being identified as chondrocytes, wherein the left image is 100X, and the right image is 400X;
图5为免疫组织化学染色后不同Pfirrmann分级的人椎间盘髓核组织中CITED4+表达的软骨细胞存在差异的结果图，100X；Figure 5 is a graph showing differences in CITED4+-expressing chondrocytes in nucleus pulposus tissues of human intervertebral discs at different Pfirrmann grades after immunohistochemical staining, 100X;
图6为在训练集中，不同组别人椎间盘髓核组织中CITED4+细胞个数、METRN+细胞积分光密度差异统计结果图，其中，A图：CITED4+细胞，B图：METRN+细胞；Figure 6 is a statistical diagram showing the difference in the number of CITED4+ cells and the integrated optical density of METRN+ cells in the nucleus pulposus tissue of intervertebral discs of different groups in the training set, wherein Figure A: CITED4+ cells, Figure B: METRN+ cells;
图7为在训练集中，CITED4+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 7 is a ROC curve obtained by plotting CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the training set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图8为在训练集中，METRN+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 8 is a ROC curve diagram obtained by plotting METRN+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the training set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图9为在训练集中，METRN+CITED4+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 9 is a ROC curve obtained by plotting METRN+CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the training set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图10为在验证集中，不同组别人椎间盘髓核组织中CITED4+细胞个数、METRN+细胞积分光密度差异统计结果图，其中，A图：CITED4+细胞，B图：METRN+细胞；Figure 10 is a graph showing the statistical results of the difference in the number of CITED4+ cells and the integrated optical density of METRN+ cells in the nucleus pulposus tissue of intervertebral discs of different groups in the validation set, wherein Figure A: CITED4+ cells, Figure B: METRN+ cells;
图11为在验证集中，CITED4+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 11 is a ROC curve obtained by plotting CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the validation set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图12为在验证集中，METRN+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 12 is a ROC curve diagram obtained by plotting METRN+ cells against different degenerative human intervertebral disc nucleus pulposus pathological tissues in the validation set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图13为在验证集中，METRN+CITED4+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 13 is a ROC curve obtained by plotting METRN+CITED4+ cells against different degenerative human intervertebral disc nucleus pulposus pathological tissues in the validation set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图14为不同组别人椎间盘髓核组织中CITED4+细胞个数差异统计结果图，其中，L：轻度退变，n＝17；M：中度退变，n＝18；S：重度退变，n＝15；LM：轻中度退变，n＝35；MS：中重度退变，n＝33；*p<0.05，**p<0.01，***p<0.001，N.S.p>0.05；Figure 14 is a statistical result of the difference in the number of CITED4+ cells in the nucleus pulposus tissue of the intervertebral disc of different groups, where L: mild degeneration, n = 17; M: moderate degeneration, n = 18; S: severe degeneration, n = 15; LM: mild to moderate degeneration, n = 35; MS: moderate to severe degeneration, n = 33; *p < 0.05, **p < 0.01, ***p < 0.001, N.S.p > 0.05;
图15为CITED4+细胞对不同退变的人椎间盘髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；Figure 15 is a ROC curve diagram obtained by plotting CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图16为免疫组织化学染色后不同Pfirrmann分级的椎间盘髓核组织中METRN+表达的软骨细胞存在差异的结果图，100X；FIG. 16 is a graph showing the difference in the expression of METRN+ chondrocytes in the nucleus pulposus tissue of the intervertebral disc with different Pfirrmann grades after immunohistochemical staining, 100X;
图17为不同组别人椎间盘髓核组织中METRN+细胞积分光密度差异统计结果图，其中，L：轻度退变，n＝17；M：中度退变，n＝18；S：重度退变，n＝15；LM：轻中度退变，n＝35；MS：中重度退变，n＝33，*p<0.05，**p<0.01，***p<0.001，N.S.p>0.05；Figure 17 is a statistical result of the difference in integrated optical density of METRN+ cells in the nucleus pulposus tissue of different groups of human intervertebral discs, where L: mild degeneration, n = 17; M: moderate degeneration, n = 18; S: severe degeneration, n = 15; LM: mild to moderate degeneration, n = 35; MS: moderate to severe degeneration, n = 33, *p < 0.05, **p < 0.01, ***p < 0.001, N.S.p> 0.05;
图18为METRN+细胞对不同退变的髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)；FIG18 is a ROC curve diagram obtained by plotting METRN+ cells for different degenerative nucleus pulposus pathological tissues, wherein, FIGA: L/M (comparison between mild degeneration and moderate degeneration), FIGB: M/S (comparison between moderate degeneration and severe degeneration), FIGC: L/S (comparison between mild degeneration and severe degeneration), FIGD: LM/S (comparison between mild-moderate degeneration and severe degeneration), FIGE: L/MS (comparison between mild degeneration and moderate-severe degeneration);
图19为METRN+CITED4+细胞对不同退变的髓核病理组织绘制得到的ROC曲线图，其中，A图：L/M(轻度退变与中度退变比较)，B图：M/S(中度退变与重度退变比较)，C图：L/S(轻度退变与重度退变比较)，D图：LM/S(轻中度退变与重度退变比较)，E图：L/MS(轻度退变与中重度退变比较)。Figure 19 is a ROC curve diagram obtained by plotting METRN+CITED4+ cells on different degenerated nucleus pulposus pathological tissues, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild to moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate to severe degeneration).
具体实施方式DETAILED DESCRIPTION本发明经过广泛而深入的研究，首次发现CITED4、METRN在不同退变程度的椎间盘退变组织中存在显著差异表达，其与椎间盘退变的程度具有极高的关联度，在本发明收集的临床样本中表现出对椎间盘退变程度的较高的鉴别诊断效能，准确性、灵敏度和特异性均较高，能够作为鉴别诊断椎间盘退变程度的生物标志物应用于临床。After extensive and in-depth research, the present invention discovered for the first time that CITED4 and METRN were significantly differentially expressed in degenerative disc tissues of different degrees of degeneration, and that they were highly correlated with the degree of disc degeneration. In the clinical samples collected by the present invention, they showed a high differential diagnostic efficacy for the degree of disc degeneration, with high accuracy, sensitivity and specificity, and can be used in clinical practice as biomarkers for differential diagnosis of the degree of disc degeneration.
除非另有说明，本文中涉及的技术术语通常按照其常规用法使用。为了进一步阐述本发明，此处对本发明涉及的部分术语进行如下解释：Unless otherwise specified, the technical terms involved in this article are generally used according to their conventional usage. In order to further explain the present invention, some of the terms involved in the present invention are explained as follows:
本文使用的术语“包括”或“包含”，是指包括所陈述的元件或组成部分中的任意一种或多种，而并未排除其它元件或其它组成部分。The terms “include” or “comprising” used herein refer to including any one or more of the stated elements or components but not excluding other elements or components.
本文使用的术语“生物标志物”，是指患者(所述患者在本发明中具体是指椎间盘退行性病变患者)表型的指示物，例如病理学状态或对治疗剂的可能的反应性的指示物，其可以在所述患者来源的生物样本中检测到，所述生物标志物包括但不限于：DNA、RNA、蛋白质、小分子代谢物质、糖类、基于糖脂的分子等；在本发明的具体实施方案中，所述生物标志物为CITED4和/或METRN。The term "biomarker" used in this article refers to an indicator of the phenotype of a patient (the patient in the present invention specifically refers to a patient with degenerative disc disease), such as an indicator of a pathological state or possible responsiveness to a therapeutic agent, which can be detected in a biological sample derived from the patient. The biomarker includes but is not limited to: DNA, RNA, protein, small molecule metabolites, carbohydrates, glycolipid-based molecules, etc.; in a specific embodiment of the present invention, the biomarker is CITED4 and/or METRN.
本文使用的术语“表达水平”同“水平”，是指本发明中所述生物标志物CITED4和/或METRN的绝对量或相对量，可以通过多种技术确定本发明中所述生物标志物CITED4和/或METRN的表达水平，特别地，可以通过使用本领域技术人员熟知的方法对本发明中所述生物标志物CITED4、METRN的绝对量或相对量进行检测。The term "expression level" used in this article is the same as "level", which refers to the absolute amount or relative amount of the biomarkers CITED4 and/or METRN described in the present invention. The expression level of the biomarkers CITED4 and/or METRN described in the present invention can be determined by a variety of techniques. In particular, the absolute amount or relative amount of the biomarkers CITED4 and METRN described in the present invention can be detected by using methods well known to those skilled in the art.
在一些实施方案中，可通过本领域熟知的多种技术来测量基因(即所述生物标志物CITED4和/或METRN)的表达水平，通常，基因的表达水平可以通过测定mRNA的量来确定。测定mRNA量的方法是本领域所熟知的。例如，样本(例如，从患者身上提取的血液、细胞或组织样本)中包含的核酸，首先根据标准化方法提取，例如使用细胞酶或化学溶液或者根据制造商的说明书通过核酸结合树脂提取。然后通过杂交(例如Northern印迹分析、原位杂交)和/或扩增(例如RT-PCR)来检测提取的mRNA。其他扩增方法包括但不限于：连接酶链式反应(LCR)、转录介导的扩增(TMA)、链置换扩增(SDA)、基于核酸序列的扩增(NASBA)。In some embodiments, the expression level of a gene (i.e., the biomarker CITED4 and/or METRN) can be measured by a variety of techniques well known in the art. Generally, the expression level of a gene can be determined by measuring the amount of mRNA. Methods for measuring the amount of mRNA are well known in the art. For example, nucleic acids contained in a sample (e.g., blood, cell or tissue sample extracted from a patient) are first extracted according to a standardized method, such as using a cell enzyme or chemical solution or extracted by a nucleic acid binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR). Other amplification methods include, but are not limited to, ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).
本文使用的术语“显著差异”，是指与第二种样品中相同的一种或多种本发明所述的生物标志物的表达水平比较，经测定所述生物标志物的含量或浓度，在一个样品中本发明的一种或多种生物标志物的含量或浓度的差异。本文所用“显著差异基因”可以用给定生物标志物(基因)的水平相对于对照中给定生物标志物的平均水平的比率进行测定，其中比率不等于1.0。还可以用p值测定差异。使用p值时，当p值小于0.1时生物标志物被鉴定为在第一和第二群体之间呈现差异。更优选p值小于0.05。甚至更优选p值小于0.01。还更优选p值小于0.005。最优选p值小于0.001。当基于比率确定差异时，如果第一种和第二种样品中水平的比率大于或小于1.0，则所述生物标志物是呈现差异的。举例来说，大于1.2、1.5、1.7、2、3、4、10、20的比率，或小于1的比率，例如0.8、0.6、0.4、0.2、0.1、0.05。The term "significant difference" as used herein refers to the difference in the content or concentration of one or more biomarkers of the present invention in one sample, compared to the expression level of the same one or more biomarkers of the present invention in a second sample, after measuring the content or concentration of the biomarkers. The "significant difference gene" used herein can be measured by the ratio of the level of a given biomarker (gene) relative to the average level of a given biomarker in a control, wherein the ratio is not equal to 1.0. The difference can also be measured using a p-value. When using a p-value, a biomarker is identified as showing a difference between the first and second populations when the p-value is less than 0.1. More preferably, the p-value is less than 0.05. Even more preferably, the p-value is less than 0.01. Still more preferably, the p-value is less than 0.005. Most preferably, the p-value is less than 0.001. When the difference is determined based on a ratio, if the ratio of the levels in the first and second samples is greater than or less than 1.0, the biomarker is different. For example, greater than 1.2, 1.5, A ratio of 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.
本文使用的术语“诊断”，是指基于与个体相关的一个或多个的症状、数据或其他信息，来对个体的健康状态或状况的发现、判断或认知。个体的健康状态可被诊断为健康的/正常的(即不存在疾病或疾患)，或者可被诊断为不健康的/异常的(即存在疾病或疾患)，或者可被诊断为具体的患病严重程度(例如轻度、轻中度、中度、中重度、重度)，术语诊断、早期诊断、进行诊断及这些术语的变化型包括与特定疾病或疾患(在本发明中具体指椎间盘退行性病变患者)相关的疾病/病症的早期发现；疾病的特性或分类；疾病的进展、治愈或复发的发现；个体的处置或治疗后对疾病的反应的发现。The term "diagnosis" as used herein refers to the discovery, judgment or recognition of an individual's health status or condition based on one or more symptoms, data or other information related to the individual. The individual's health status can be diagnosed as healthy/normal (i.e., the absence of disease or illness), or can be diagnosed as unhealthy/abnormal (i.e., the presence of disease or illness), or can be diagnosed as a specific severity of illness (e.g., mild, mild-moderate, moderate, moderate-severe, severe). The terms diagnosis, early diagnosis, diagnosis and variations of these terms include early discovery of diseases/disorders associated with specific diseases or illnesses (specifically, patients with degenerative disc disease in the present invention); characteristics or classification of the disease; discovery of the progression, cure or recurrence of the disease; discovery of the individual's response to the disease after treatment or treatment.
在一些实施方案中，本发明提供的检测探针还可包含一种或多种可检测标记物，所述可检测标记物的具体实例包括荧光分子(或荧光染料)、荧光纳米微粒、放射性同位素、可检测酶等。在一些实施方案中，所述荧光分子可从《使用手册--荧光探针和标记技术指南》中选择，可以结合到核酸分子的特殊荧光基团包括但不限于：4-乙酰氨基-4’-异硫氰酸二苯乙烯-2,2’-二磺酸、吖啶、异硫氰酸酯吖啶、5-(2’-氨乙基)氨基萘-1-磺酸、4-氨基-N-[3-乙烯基磺酰基)苯基]萘酰亚胺-3,5-二磺酸盐、N-(4-苯胺基-1-萘基)马来酰亚胺、邻氨基苯甲酰胺、亮黄、香豆素及其衍生物等。在一些实施方案中，所述荧光纳米微粒包括但不限于：半导体纳米晶体、量子点等。在一些实施方案中，所述放射性同位素包括但不限于：放射性碘、放射性铯、放射性铱、放射性钴等。在一些实施方案中，所述可检测酶包括但不限于：辣根过氧化物酶、碱性磷酸酶、酸性磷酸酶、葡萄糖氧化酶、β-半乳糖苷酶、β-葡萄糖醛酸酶、β-内酰胺酶等。In some embodiments, the detection probe provided by the present invention may also include one or more detectable markers, and specific examples of the detectable markers include fluorescent molecules (or fluorescent dyes), fluorescent nanoparticles, radioactive isotopes, detectable enzymes, etc. In some embodiments, the fluorescent molecules can be selected from the User Manual - Fluorescent Probes and Labeling Technology Guide, and special fluorescent groups that can be bound to nucleic acid molecules include but are not limited to: 4-acetylamino-4'-isothiocyanate stilbene-2,2'-disulfonic acid, acridine, isothiocyanate acridine, 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid, 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate, N-(4-anilino-1-naphthyl)maleimide, o-aminobenzamide, brilliant yellow, coumarin and its derivatives, etc. In some embodiments, the fluorescent nanoparticles include but are not limited to: semiconductor nanocrystals, quantum dots, etc. In some embodiments, the radioactive isotopes include but are not limited to: radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt, etc. In some embodiments, the detectable enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, β-lactamase, and the like.
本文使用的术语“曲线下面积”或“AUC”，是指受试者工作特征(ROC)曲线的曲线下面积，二者是本领域所熟知的。AUC测量对于跨全部数据范围比较分类器的准确度是有用的。具有较高AUC的分类器具有在两个或多个目标组(例如，不同退变程度的椎间盘退行性病变患者的椎间盘髓核组织样品)之间进行正确分类未知的更高的能力。ROC曲线对于在两个群体之间进行区别时描绘特定特征(例如，本文所描述的任何生物标志物和/或另外的生物医学信息的任何条目)的性能是有用的。通常，以单个特征的值为基础以升序顺序跨越整个群体选出特征数据。然后，对于该特征的每个值，计算数据的真阳性率和假阳性率。真阳性率通过计数高于该特征的值的病例的数目并除以病例总数而确定。假阳性率通过计数高于该特征的值的对照的数目并除以对照总数而确定。ROC曲线可关于单独特征来生成，且可关于其他单独输出来生成，例如，两个或更多个特征的组合可用数学方法结合(例如，相加、相减、相乘等)以提供单独的总和值，且该单独的总和值可绘制于ROC曲线中。另外，其中的组合源自单独的输出值的多个特征的任意组合可绘制于ROC曲线中。特征的这些组合可包括试验。ROC曲线是试验的真阳性率(敏感性)针对试验的假阳性率(1-特异性)的图，能够用于分析诊断的准确性。The term "area under the curve" or "AUC" as used herein refers to the area under the curve of the receiver operating characteristic (ROC) curve, both of which are well known in the art. The AUC measurement is useful for comparing the accuracy of classifiers across the entire data range. A classifiers with higher AUC have a higher ability to correctly classify unknowns between two or more target groups (e.g., intervertebral disc nucleus pulposus tissue samples of patients with intervertebral disc degeneration of different degrees of degeneration). The ROC curve is useful for describing the performance of a specific feature (e.g., any biomarker described herein and/or any item of additional biomedical information) when distinguishing between two populations. Typically, feature data is selected across the entire population in ascending order based on the value of a single feature. Then, for each value of the feature, the true positive rate and false positive rate of the data are calculated. The true positive rate is determined by counting the number of cases with a value higher than the feature and divided by the total number of cases. The false positive rate is determined by counting the number of controls with a value higher than the feature and divided by the total number of controls. ROC curves can be generated with respect to individual features, and can be generated with respect to other individual outputs, for example, a combination of two or more features can be combined mathematically (e.g., added, subtracted, multiplied, etc.) to provide a separate sum value, and the separate sum value can be plotted in the ROC curve. In addition, any combination of multiple features in which the combination is derived from a separate output value can be plotted in the ROC curve. These combinations of features can include tests. The ROC curve is a plot of the true positive rate (sensitivity) of a test against the false positive rate (1-specificity) of the test, which can be used to analyze the accuracy of diagnosis.
本文使用的术语“敏感性”，是指相对于目标患者总数量(100％)的真阳性患者数量(％)，敏感性通过以下公式计算：敏感性＝TP/(TP+FN)(TP＝真阳性；FN＝假阴性)。The term "sensitivity" used in this article refers to the number of true positive patients (%) relative to the total number of target patients (100%), and sensitivity is calculated by the following formula: Sensitivity = TP/(TP+FN) (TP = true positive; FN = false negative).
本文使用的术语“特异性”，是指相对于健康受试者总数量(100％)的真阴性个体数量(％)，特异性通过以下公式计算：特异性＝TN/(TN+FP)(TN＝真阴性；FP＝假阳性)。The term "specificity" used in this article refers to the number of true negative individuals (%) relative to the total number of healthy subjects (100%), and the specificity is calculated by the following formula: Specificity = TN/(TN+FP) (TN = true negative; FP = false positive).
本文使用的术语“样本”或“样品”，是指获自或衍生自目标受试者的组合物，其包含有待例如基于物理、生物化学、化学和/或生理学特征表征和/或鉴定的细胞实体和/或其他分子实体。该样本可以获自受试者的血液和生物来源的其他流体样本及组织样本，如活检组织样本或从其衍生的组织培养物或细胞。组织样本的来源可以是实体组织，如来自新鲜、冷冻和/或保藏的器官或组织样本、活检组织或吸出物；血液或任意血液组分；体液；来自个体妊娠或发育的任何时间的细胞；或血浆。术语样本包括在其获得后以任何方式处理过的生物样本，如经试剂处理、稳定化、或针对某些成分(如蛋白质或多核苷酸)富集、或包埋在用于切片目的的半固体或固体基质中。本发明中所述的样本或样品包括但不限于：组织样本、血液样本、血液来源的细胞样本、血清样本、血浆样本、淋巴液样本、滑膜液样本、细胞提取物样本或其任意组合。The term "sample" or "sample" as used herein refers to a composition obtained from or derived from a target subject, which contains cell entities and/or other molecular entities to be characterized and/or identified, for example, based on physical, biochemical, chemical and/or physiological characteristics. The sample can be obtained from the subject's blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be a solid tissue, such as an organ or tissue sample, a biopsy tissue or an aspirate from a fresh, frozen and/or preserved organ; blood or any blood component; body fluid; cells from any time of pregnancy or development of an individual; or plasma. The term sample includes biological samples that have been processed in any way after they are obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. The sample or sample described in the present invention includes, but is not limited to: a tissue sample, a blood sample, a blood-derived cell sample, a serum sample, a plasma sample, a lymph sample, a synovial fluid sample, a cell extract sample, or any combination thereof.
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测。The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It should be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.
实施例1单细胞转录组测序分析实验Example 1 Single cell transcriptome sequencing analysis experiment
1、实验材料1. Experimental Materials
人退变椎间盘髓核组织5例，ChromiumTM Single Cell 3'Solution微流体平台，10X Genomics单细胞测序平台。5 cases of human degenerative intervertebral disc nucleus pulposus tissue, ChromiumTM Single Cell 3'Solution microfluidic platform, 10X Genomics single-cell sequencing platform.
2、实验方法2. Experimental methods
(1)收集人椎间盘髓核组织样本(1) Collection of human intervertebral disc nucleus pulposus tissue samples
收集人退变椎间盘髓核组织5例。样本为根据临床实际诊疗情况，必须行手术摘除治疗外伤导致腰椎爆裂性骨折患者或退行性腰椎间盘突出患者所必要留取的椎间盘病理组织，本研究获得医院医学伦理委员会的批准和所述受试者或受试者家属(监护人)的知情同意。Five cases of human degenerative intervertebral disc nucleus pulposus tissue were collected. The samples were pathological tissues of intervertebral discs that must be removed surgically for patients with lumbar burst fractures or degenerative lumbar disc herniation caused by trauma according to actual clinical diagnosis and treatment. This study was approved by the hospital's medical ethics committee and the informed consent of the subjects or their family members (guardians).
(2)获取椎间盘髓核组织的软骨细胞并制备单细胞悬液(2) Obtaining chondrocytes from intervertebral disc nucleus pulposus tissue and preparing single cell suspension
用PBS将髓核组织清洗干净，眼科剪将清洗干净后的髓核组织剪碎。将剪碎的组织转移到含有组织解离液的5mL离心管中，37℃条件下胰酶消化0.5h，II型胶原酶37℃条件下旋转消化2.5-3h，通过70μm细胞筛滤膜，孵育结束后，裂解红细胞，通过40μm细胞筛滤膜，去除死细胞，4℃条件下梯度离心，PBS重悬细胞至适当的体积。The nucleus pulposus tissue was cleaned with PBS and minced with ophthalmic scissors. The minced tissue was transferred to a 5 mL centrifuge tube containing tissue dissociation solution, digested with trypsin at 37°C for 0.5 h, and digested with type II collagenase at 37°C for 2.5-3 h, and passed through a 70 μm cell sieve membrane. After the incubation, the red blood cells were lysed, and the dead cells were removed by passing through a 40 μm cell sieve membrane, and gradient centrifuged at 4°C, and the cells were resuspended in PBS to an appropriate volume.
(3)上机测序(3) Sequencing
采用基于10X Genomics平台的ChromiumTM Single Cell 3'Solution一次性分离、并标记500-10000范围内的所有单细胞，在单细胞水平进行基因表达检测。具体流程是ChromiumTM Single Cell 3'Solution是建立在GemCode技术上的微流体平台，将带有条形码和引物的凝胶珠和单个细胞包裹在油滴中；接下来在每个油滴内，凝胶珠溶解，细胞裂解释放mRNA，通过逆转录产生用于测序的带条形码的cDNA。液体油层破坏后，cDNA后续进行文库构建，然后使用Illumina测序平台对文库进行测序检测，一次性获得大量单细胞的基因表达数据，从而实现在单细胞水平进行表达测序。The ChromiumTM Single Cell 3'Solution based on the 10X Genomics platform is used to isolate and label all single cells in the range of 500-10,000 at one time, and perform gene expression detection at the single-cell level. The specific process is that the ChromiumTM Single Cell 3'Solution is a microfluidic platform based on the GemCode technology, which encapsulates gel beads with barcodes and primers and single cells in oil droplets; then in each oil droplet, the gel beads are dissolved, the cells are lysed to release mRNA, and reverse transcription is used to produce barcoded cDNA for sequencing. After the liquid oil layer is destroyed, the cDNA is subsequently used to construct a library, and then the library is sequenced and detected using the Illumina sequencing platform, obtaining a large amount of single-cell gene expression data at one time, thereby realizing expression sequencing at the single-cell level.
(4)数据处理和细胞亚群的鉴定(4) Data processing and identification of cell subpopulations
分别对所有样本进行分群分析，鉴定细胞亚群，发现罕见细胞亚群。对实验组和对照组数据进行合并，进行分群分析。比较两组样品在细胞分群中的不同分布以及不同细胞类型的差异占比情况。进行差异基因表达分析，差异分析是基于细胞分群结果进行的，目的是找到每个亚群的显著差异基因，作为亚群的特征基因，从而协助识别细胞类型。通过差异基因功能注释和富集分析，即GO注释富集分析、KEGG注释富集分析、疾病注释富集分析、转录因子注释、蛋白互作网络，协助识别细胞的功能。通过以上技术对人椎间盘髓核细胞进行亚群识别。All samples were clustered and analyzed separately to identify cell subpopulations and find rare cell subpopulations. The data of the experimental group and the control group were merged for cluster analysis. The different distributions of the two groups of samples in the cell clusters and the different proportions of different cell types were compared. Differential gene expression analysis was performed. The differential analysis was based on the results of cell clustering. The purpose was to find significantly differential genes in each subpopulation as characteristic genes of the subpopulation, thereby assisting in identifying cell types. Differential gene function annotation and enrichment analysis, namely GO annotation enrichment analysis, KEGG annotation enrichment analysis, disease annotation enrichment analysis, transcription factor annotation, and protein interaction network, were used to assist in identifying cell functions. The above techniques were used to identify subpopulations of human intervertebral disc nucleus pulposus cells.
3、实验结果3. Experimental results
在单细胞测序上机前，对制备的单细胞悬液进行质控，以其中1例人椎间盘髓核组织样本为例，结果见图1，细胞活率为84.17％，细胞结团率为26.09％，活细胞浓度为730cells/μL，细胞平均直径为12.26μm，活细胞颗粒比为77.05％。Before single-cell sequencing, the prepared single-cell suspension was quality controlled. Taking a human intervertebral disc nucleus pulposus tissue sample as an example, the results are shown in Figure 1. The cell viability was 84.17%, the cell agglomeration rate was 26.09%, the live cell concentration was 730 cells/μL, the average cell diameter was 12.26 μm, and the live cell particle ratio was 77.05%.
通过采用基于10X Genomics平台的ChromiumTM Single Cell 3'Solution对人椎间盘髓核组织单细胞进行分离，在单细胞水平进行基因表达检测。通过数据处理和生物信息学分析，识别人椎间盘髓核组织单细胞亚群，结果见图2，其中，第8亚群为新发现的椎间盘髓核组织软骨细胞亚群。The ChromiumTM Single Cell 3'Solution based on the 10X Genomics platform was used to isolate single cells from human intervertebral disc nucleus pulposus tissue, and gene expression was detected at the single cell level. Through data processing and bioinformatics analysis, single cell subpopulations of human intervertebral disc nucleus pulposus tissue were identified, and the results are shown in Figure 2. Among them, subpopulation 8 is a newly discovered subpopulation of chondrocytes in intervertebral disc nucleus pulposus tissue.
为了进一步准确识别第8亚群，本实施例对该亚群的差异基因进行分析，结果见图3，结果显示CITED4和METRN为这种新发现的椎间盘髓核组织软骨细胞亚群的特征基因。In order to further accurately identify the 8th subgroup, the differential genes of this subgroup were analyzed in this example. The results are shown in FIG3 . The results show that CITED4 and METRN are the characteristic genes of this newly discovered subgroup of chondrocytes in the nucleus pulposus tissue of the intervertebral disc.
实施例2椎间盘髓核组织MRI分级及样本分组Example 2 MRI grading of intervertebral disc nucleus pulposus tissue and sample grouping
1、实验材料1. Experimental Materials
人退变椎间盘组织50例。50 cases of human degenerative disc tissue.
2、实验方法2. Experimental methods
(1)样本收集(1) Sample collection
收集人退变椎间盘组织50例。样本为根据临床实际诊疗情况，必须行手术摘除治疗外伤导致腰椎爆裂性骨折患者或退行性腰椎间盘突出患者所留取的退变椎间盘病理组织，本研究获得医院医学伦理委员会的批准和所述受试者或受试者家属(监护人)的知情同意。50 cases of human degenerative intervertebral disc tissue were collected. The samples were pathological tissues of degenerative intervertebral discs from patients with lumbar burst fractures or degenerative lumbar disc herniation caused by trauma, which had to be surgically removed according to the actual clinical diagnosis and treatment. This study was approved by the hospital's medical ethics committee and the informed consent of the subjects or their family members (guardians).
(2)MRI(核磁共振成像)技术鉴定不同椎间盘组织的退变程度(2) MRI (magnetic resonance imaging) technology to identify the degree of degeneration of different intervertebral disc tissues
采用Pfirrmann椎间盘退变的MRI(T2WI)分级标准对所有患者椎间盘退变的程度进行分级。I级：椎间盘结构质均、色亮白，髓核与纤维环边界清，信号强度高或等于脑脊液，椎间盘高度正常。II级：椎间盘结构非均质、有或无水平带，髓核与纤维环边界清，信号强度高或等于脑脊液，椎间盘高度正常。III级：椎间盘结构非均质、灰，髓核与纤维环边界不清，信号强度中等，椎间盘高度正常或轻度降低。IV级：椎间盘结构非均质、灰或黑，髓核与纤维环边界消失，信号强度中等或低信号，椎间盘高度正常或中度降低。V级：椎间盘结构非均质、黑，髓核与纤维环边界消失，信号强度低信号，椎间盘间隙塌陷。The degree of disc degeneration in all patients was graded using the Pfirrmann MRI (T2WI) grading standard for disc degeneration. Grade I: The disc structure is homogeneous and bright white, the boundary between the nucleus pulposus and the annulus fibrosus is clear, the signal intensity is high or equal to the cerebrospinal fluid, and the disc height is normal. Grade II: The disc structure is heterogeneous, with or without horizontal bands, the boundary between the nucleus pulposus and the annulus fibrosus is clear, the signal intensity is high or equal to the cerebrospinal fluid, and the disc height is normal. Grade III: The disc structure is heterogeneous and gray, the boundary between the nucleus pulposus and the annulus fibrosus is unclear, the signal intensity is medium, and the disc height is normal or slightly reduced. Grade IV: The disc structure is heterogeneous, gray or black, the boundary between the nucleus pulposus and the annulus fibrosus disappears, the signal intensity is medium or low, and the disc height is normal or moderately reduced. Grade V: The disc structure is heterogeneous, gray or black, the boundary between the nucleus pulposus and the annulus fibrosus disappears, the signal intensity is medium or low, and the disc height is normal or moderately reduced. The disc structure is heterogeneous and black, the boundary between the nucleus pulposus and the annulus fibrosus disappears, the signal intensity is low, and the intervertebral disc space is collapsed.
(3)样本分组(3) Sample grouping
将椎间盘组织根据MRI结果进行Pfirrmann分级后，结果I～II级分为轻度退行性病变组(L)，III为中度退行性病变组(M)，IV～V级分为重度退行性病变组(S)。此外，为了区别该生物标志物随着退变严重程度而存在的表达差异，从而更好的评价CITED4和METRN作为生物标志物的诊断效能，将I～II和III进一步合并为轻中度退行性病变组(LM)，III和IV～V级分为中重度退行性病变组(MS)。After the intervertebral disc tissue was graded by Pfirrmann according to the MRI results, the results showed that grades I to II were divided into mild degenerative lesions group (L), III into moderate degenerative lesions group (M), and IV to V into severe degenerative lesions group (S). In addition, in order to distinguish the expression differences of the biomarker with the severity of degeneration, so as to better evaluate the diagnostic efficacy of CITED4 and METRN as biomarkers, grades I to II and III were further combined into mild to moderate degenerative lesions group (LM), and grades III and IV to V were divided into moderate to severe degenerative lesions group (MS).
3、实验结果3. Experimental results
50例人椎间盘退变组织的Pfirrmann退变分级结果见下表1，结果显示Pfirrmann退变分级I～II(轻度，L)样本17例，III(中度，M)样本18例，IV～V(重度，S)样本15例。进一步分组得到I～III(轻中度，LM)样本共35例，III～V(中重度，MS)样本共33例。The Pfirrmann degeneration grading results of 50 cases of human intervertebral disc degeneration tissues are shown in Table 1 below. The results show that there are 17 samples with Pfirrmann degeneration grading I-II (mild, L), 18 samples with III (moderate, M), and 15 samples with IV-V (severe, S). Further grouping resulted in 35 samples with I-III (mild-moderate, LM) and 33 samples with III-V (moderate-severe, MS).
表1人椎间盘退变组织的Pfirrmann退变分级
Table 1 Pfirrmann degeneration grading of human intervertebral disc degeneration tissue
实施例3 ACAN软骨细胞鉴定实验Example 3 ACAN chondrocyte identification experiment
1、实验材料1. Experimental Materials
人退变椎间盘组织50例，Anti-Aggrecan Antibody(ACAN)(博士德生物，BA2967-1，Lot No:D-blj2-08F183)。50 cases of human degenerative intervertebral disc tissue, Anti-Aggrecan Antibody (ACAN) (Boster Biotech, BA2967-1, Lot No: D-blj2-08F183).
2、实验方法2. Experimental methods
(1)样本收集(1) Sample collection
收集实施例2中经MRI鉴定过的人退变椎间盘组织50例。样本为根据临床实际诊疗情况，必须行手术摘除治疗外伤导致腰椎爆裂性骨折患者或退行性腰椎间盘突出患者所留取的退变椎间盘病理组织，本研究获得医院医学伦理委员会的批准和所述受试者或受试者家属(监护人)的知情同意。50 cases of human degenerative intervertebral disc tissue identified by MRI in Example 2 were collected. The samples were pathological tissues of degenerative intervertebral discs collected from patients with lumbar burst fractures or degenerative lumbar disc herniation caused by trauma, which must be surgically removed according to actual clinical diagnosis and treatment. This study was approved by the hospital's medical ethics committee and the informed consent of the subjects or their family members (guardians).
(2)免疫组化染色及判读(2) Immunohistochemical staining and interpretation
常规组织固定、脱水、包埋、石蜡切片，65℃烤片1h，脱蜡、水化，酶修复法进行抗原修复，细胞样本用95％乙醇充分固定，3％过氧化氢孵育10min，PBS洗2次，一抗孵育30min，Anti-Aggrecan Antibody稀释浓度为1：50，PBS洗2次，二抗孵育20min，PBS洗2次，DAB染色，自来水充分冲洗，复染，脱水，透明，封片。Routine tissue fixation, dehydration, embedding, paraffin sectioning, baking at 65℃ for 1h, dewaxing, hydration, antigen retrieval by enzyme retrieval method, cell samples fully fixed with 95% ethanol, incubated with 3% hydrogen peroxide for 10min, washed twice with PBS, incubated with primary antibody for 30min, Anti-Aggrecan Antibody dilution concentration of 1:50, washed twice with PBS, incubated with secondary antibody for 20min, washed twice with PBS, DAB staining, fully rinsed with tap water, counterstained, dehydrated, transparent, and sealed.
切片的病理学结果由两名病理学家独立复查，并使用共识解释作为最终的结果。不同的结果由第三位病理学家裁决。Pathological results of the slides were reviewed independently by two pathologists, and the consensus interpretation was used as the final result. Disagreeing results were adjudicated by a third pathologist.
3、实验结果3. Experimental results
软骨蛋白聚糖Aggrecan(ACAN)是软骨细胞外基质的重要成分，通常作为标志物用于鉴定软骨细胞。本实施例的鉴定结果见图4，通过免疫组化技术用Aggrecan(ACAN)抗体鉴定人椎间盘髓核组织中表达阳性的细胞即为软骨细胞。Cartilage proteoglycan Aggrecan (ACAN) is an important component of the extracellular matrix of cartilage cells and is usually used as a marker to identify chondrocytes. The identification results of this example are shown in Figure 4. Cells expressing positive in human intervertebral disc nucleus pulposus tissue identified by immunohistochemistry using Aggrecan (ACAN) antibody are chondrocytes.
实施例4 CITED4、METRN免疫组化实验及ROC曲线分析Example 4 CITED4, METRN immunohistochemistry experiment and ROC curve analysis
1、实验材料1. Experimental Materials
人退变椎间盘组织50例，Anti-CITED4antibody(美国abcam公司，ab134072，YJ082412CS)，Meteorin Polyclonal Antibody(美国Invitrogen公司，PA5-62335，XF3619498A)。50 samples of human degenerative intervertebral disc tissues, Anti-CITED4antibody (abcam, USA, ab134072, YJ082412CS), Meteorin Polyclonal Antibody (Invitrogen, USA, PA5-62335, XF3619498A).
2、实验方法2. Experimental methods
(1)样本收集(1) Sample collection
收集实施例2中经MRI鉴定过的人退变椎间盘组织50例。样本为根据临床实际诊疗情况，必须行手术摘除治疗外伤导致腰椎爆裂性骨折患者或退行性腰椎间盘突出患者所留取的病变椎间盘病理组织，本研究获得医院医学伦理委员会的批准和所述受试者或受试者家属(监护人)的知情同意。50 cases of human degenerative intervertebral disc tissue identified by MRI in Example 2 were collected. The samples were pathological tissues of diseased intervertebral discs obtained from patients with lumbar burst fractures or degenerative lumbar disc herniation caused by trauma, which must be surgically removed according to actual clinical diagnosis and treatment. This study was approved by the hospital's medical ethics committee and the informed consent of the subjects or their family members (guardians).
(2)免疫组化染色及判读(2) Immunohistochemical staining and interpretation
常规组织固定、脱水、包埋、石蜡切片，65℃烤片1h，脱蜡、水化，酶修复法进行抗原修复，细胞样本95％乙醇充分固定，3％过氧化氢孵育10min，PBS洗2次，一抗孵育30min，Anti-CITED4antibody和Meteorin Polyclonal Antibody稀释比例分别为1：100和1：50，PBS洗2次，二抗孵育20min，PBS洗2次，DAB染色，自来水充分冲洗，复染，脱水，透明，封片。Routine tissue fixation, dehydration, embedding, paraffin sectioning, baking at 65℃ for 1h, dewaxing, hydration, antigen retrieval by enzyme retrieval method, cell samples fully fixed with 95% ethanol, incubated with 3% hydrogen peroxide for 10min, washed twice with PBS, incubated with primary antibody for 30min, the dilution ratio of Anti-CITED4antibody and Meteorin Polyclonal Antibody was 1:100 and 1:50 respectively, washed twice with PBS, incubated with secondary antibody for 20min, washed twice with PBS, stained with DAB, fully rinsed with tap water, counterstained, dehydrated, transparent, and sealed.
(3)ROC曲线分析(3) ROC curve analysis
随机对每张免疫组化切片进行3个视野的随机采图，每个视野在10X镜下被拍摄。通过Image Pro Plus软件分析每张切片3个随机视野的CITED4+的阳性细胞数，将细胞总数进行统计分析；METRN+阳性表达的积分光密度采用3个随机视野的总和。病理学结果由两名病理学家独立分析，并使用共识解释作为最终的结果，不同的结果由第三位病理学家裁决。采用SPSS软件通过平均值对轻度退行性病变和中重度退行性病变绘制ROC曲线，计算AUC曲线下面积和阈值，评价新发现的软骨细胞亚群特征基因CITED4、METRN、以及CITED4和METRN联合对椎间盘退变程度的诊断价值。Three fields of view were randomly sampled for each immunohistochemical section, and each field of view was photographed under a 10X microscope. The number of CITED4+ positive cells in three random fields of view of each section was analyzed by Image Pro Plus software, and the total number of cells was statistically analyzed; the integrated optical density of METRN+ positive expression was the sum of three random fields of view. The pathological results were analyzed independently by two pathologists, and the consensus interpretation was used as the final result. Different results were adjudicated by a third pathologist. SPSS software was used to draw ROC curves for mild degenerative lesions and moderate to severe degenerative lesions by using the average value, and the area under the AUC curve and threshold were calculated to evaluate the diagnostic value of the newly discovered chondrocyte subset characteristic genes CITED4, METRN, and the combination of CITED4 and METRN for the degree of intervertebral disc degeneration.
此外，本实施例将本发明收集得到的50例人退变椎间盘组织采用R语言caret包的createDataPartition函数随机分成两组(训练集和验证集)，各占50％，也即训练集包含25例人退变椎间盘组织，验证集包含25例人退变椎间盘组织，进一步采用训练集和验证集进行了分析和验证。In addition, in this embodiment, the 50 cases of human degenerative intervertebral disc tissues collected by the present invention are randomly divided into two groups (training set and validation set) using the createDataPartition function of the caret package in the R language, each accounting for 50%, that is, the training set contains 25 cases of human degenerative intervertebral disc tissues, and the validation set contains 25 cases of human degenerative intervertebral disc tissues. The training set and the validation set are further used for analysis and verification.
3、实验结果3. Experimental results
在训练集中，CITED4和METRN在各组之间的差异表达结果见图6，结果显示CITED4和METRN在不同椎间盘退变程度的患者之间存在显著性的差异表达。In the training set, the differential expression results of CITED4 and METRN among the groups are shown in Figure 6, and the results show that CITED4 and METRN are significantly differentially expressed between patients with different degrees of intervertebral disc degeneration.
在训练集中，CITED4+对应的各组间的ROC曲线分析结果见图7和下表2。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.875，对应的特异性为77.8％，灵敏度为87.5％；轻度与重度相比(L/S)AUC值为0.931，对应的特异性为88.9％，灵敏度为87.5％；轻中度与重度(LM/S)相比AUC值为0.903，对应的特异性为83.3％，灵敏度为87.5％。结果表明CITED4是能够标记椎间盘髓核组织退变严重程度的生物标志物，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在极高的应用价值。In the training set, the ROC curve analysis results of CITED4+ between the groups are shown in Figure 7 and Table 2 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.875, the corresponding specificity is 77.8%, and the sensitivity is 87.5%; the AUC value of mild compared with severe (L/S) is 0.931, the corresponding specificity is 88.9%, and the sensitivity is 87.5%; the AUC value of mild-moderate compared with severe (LM/S) is 0.903, the corresponding specificity is 83.3%, and the sensitivity is 87.5%. The results show that CITED4 is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, which has extremely high application value.
表2在训练集中，CITED4+细胞各组别ROC曲线参数
Table 2 ROC curve parameters of CITED4+ cell groups in the training set
在训练集中，METRN+对应的各组间的ROC曲线结果见图8和下表3。除轻度与中度(L/M)和轻度与中重度(L/MS)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.806，对应的特异性为66.7％，灵敏度为100％，轻度与重度相比(L/S)AUC值为0.875，对应的特异性为77.8％，灵敏度为100％，轻中度与重度(LM/S)相比AUC值为0.840，对应的特异性为72.2％，灵敏度为100％。结果表明METRN是能够标记椎间盘髓核组织退变严重程度的生物标志物，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在极高的应用价值。In the training set, the ROC curve results of each group corresponding to METRN+ are shown in Figure 8 and Table 3 below. Except for mild to moderate (L/M) and mild to moderately severe (L/MS), the AUC values of the ROC curves of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate to severe (M/S) is 0.806, the corresponding specificity is 66.7%, and the sensitivity is 100%, the AUC value of mild to severe (L/S) is 0.875, the corresponding specificity is 77.8%, and the sensitivity is 100%, and the AUC value of mild to moderate (LM/S) is 0.840, the corresponding specificity is 72.2%, and the sensitivity is 100%. The results show that METRN is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild to moderate and severe degenerative tissues, which has extremely high application value.
表3在训练集中，METRN+细胞各组别ROC曲线参数
Table 3 ROC curve parameters of each group of METRN+ cells in the training set
在训练集中，CITED4+METRN+对应的各组间的ROC曲线结果见图9和下表4。除轻度与中度(L/M)和轻度与中重度(L/MS)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.875，对应的特异性为77.8％，灵敏度为87.5％，轻度与重度相比(L/S)AUC值为0.875，对应的特异性为100％，灵敏度为75.0％，轻中度与重度(LM/S)相比AUC值为0.917，对应的特异性为88.9％，灵敏度为87.5％。结果表明CITED4和METRN两者联合能够标记椎间盘髓核组织退变严重程度，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在显著更优的应用价值。In the training set, the ROC curve results of CITED4+METRN+ for each group are shown in Figure 9 and Table 4 below. The ROC curve AUC values of the remaining groups were greater than 0.7, except for the groups of moderate to moderate (L/M) and mild to moderately severe (L/MS). Among them, the AUC value of moderate to severe (M/S) was 0.875, with a corresponding specificity of 77.8% and a sensitivity of 87.5%, the AUC value of mild to severe (L/S) was 0.875, with a corresponding specificity of 100% and a sensitivity of 75.0%, and the AUC value of mild to moderate (LM/S) was 0.917, with a corresponding specificity of 88.9% and a sensitivity of 87.5%. The results showed that the combination of CITED4 and METRN can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in distinguishing moderate from severe, mild from severe, and mild to moderate from severe degenerative tissues, and has a significantly better application value.
表4在训练集中，CITED4+METRN+细胞各组别ROC曲线参数
Table 4 ROC curve parameters of CITED4+METRN+ cells in each group in the training set
在验证集中，CITED4和METRN在各组之间的差异表达结果见图10，结果显示CITED4和METRN在不同椎间盘退变程度的患者之间存在显著性的差异表达。In the validation set, the differential expression results of CITED4 and METRN among the groups are shown in Figure 10, and the results show that CITED4 and METRN are significantly differentially expressed between patients with different degrees of intervertebral disc degeneration.
在验证集中，CITED4+对应的各组间的ROC曲线分析结果见图11和下表5。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.968，对应的特异性为100％，灵敏度为85.7％；轻度与重度相比(L/S)AUC值为1.000，对应的特异性为100％，灵敏度为100％；轻中度与重度(LM/S)相比AUC值为1.000，对应的特异性为100％，灵敏度为100％。结果表明CITED4是能够标记椎间盘髓核组织退变严重程度的生物标志物，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在极高的应用价值。In the validation set, the ROC curve analysis results of CITED4+ between the groups are shown in Figure 11 and Table 5 below. Except for mild and moderate (L/M), the ROC curve AUC values of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.968, the corresponding specificity is 100%, and the sensitivity is 85.7%; the AUC value of mild compared with severe (L/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%; the AUC value of mild-moderate compared with severe (LM/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%. The results show that CITED4 is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, which has extremely high application value.
表5在验证集中，CITED4+细胞各组别ROC曲线参数
Table 5 ROC curve parameters of CITED4+ cell groups in the validation set
在验证集中，METRN+对应的各组间的ROC曲线结果见图12和下表6。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.984，对应的特异性为88.9％，灵敏度为100％，轻度与重度相比(L/S)AUC值为0.982，对应的特异性为87.5％，灵敏度为100％，轻中度与重度(LM/S)相比AUC值为0.983，对应的特异性为88.2％，灵敏度为100％。结果表明METRN是能够标记椎间盘髓核组织退变严重程度的生物标志物，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在极高的应用价值。In the validation set, the ROC curve results of each group corresponding to METRN+ are shown in Figure 12 and Table 6 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.984, the corresponding specificity is 88.9%, and the sensitivity is 100%, the AUC value of mild compared with severe (L/S) is 0.982, the corresponding specificity is 87.5%, and the sensitivity is 100%, and the AUC value of mild-moderate compared with severe (LM/S) is 0.983, the corresponding specificity is 88.2%, and the sensitivity is 100%. The results show that METRN is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, which has extremely high application value.
表6在验证集中，METRN+细胞各组别ROC曲线参数
Table 6 ROC curve parameters of each group of METRN+ cells in the validation set
在验证集中，CITED4+METRN+对应的各组间的ROC曲线结果见图13和下表7。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为1.000，对应的特异性为100％，灵敏度为100％，轻度与重度相比(L/S)AUC值为1.000，对应的特异性为100％，灵敏度为100％，轻中度与重度(LM/S)相比AUC值为1.000，对应的特异性为100％，灵敏度为100％。结果表明CITED4和METRN两者联合能够标记椎间盘髓核组织退变严重程度，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在显著更优的应用价值。In the validation set, the ROC curve results of CITED4+METRN+ for each group are shown in Figure 13 and Table 7 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%. The AUC value of mild compared with severe (L/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%. The AUC value of mild-moderate compared with severe (LM/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%. The results show that the combination of CITED4 and METRN can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in distinguishing between moderate and severe, mild and severe, mild-moderate and There is significantly better application value in severely degenerative tissues.
表7在验证集中，CITED4+METRN+细胞各组别ROC曲线参数
Table 7 ROC curve parameters of CITED4+METRN+ cells in each group in the validation set
在本发明收集得到的总的样本集中，CITED4在软骨细胞的核中呈阳性表达。如图5可见，随着退变严重程度增加，CITED4+的阳性细胞的数量明显增多。轻度退变的CITED4+细胞约占总细胞的3％，中度退变的CITED4+细胞约占10％，而重度退变的CITED4+细胞约为60％。组间统计学差异分析结果见图14，中度与重度相比(M/S)存在显著差异(P<0.001)，轻度与重度(L/S)相比存在显著差异(P<0.001)，轻中度与重度相比(LM/S)存在显著差异(P<0.001)，轻度与中重度相比(L/MS)存在显著差异(P＝0.002)。In the total sample set collected by the present invention, CITED4 is positively expressed in the nucleus of chondrocytes. As shown in Figure 5, as the severity of degeneration increases, the number of CITED4+ positive cells increases significantly. Mildly degenerated CITED4+ cells account for about 3% of the total cells, moderately degenerated CITED4+ cells account for about 10%, and severely degenerated CITED4+ cells account for about 60%. The results of the statistical difference analysis between the groups are shown in Figure 14. There is a significant difference (P<0.001) between moderate and severe (M/S), there is a significant difference (P<0.001) between mild and severe (L/S), there is a significant difference (P<0.001) between mild and moderate (LM/S) and severe (P<0.001), there is a significant difference (P=0.002) between mild and moderately severe (L/MS).
在本发明收集得到的总的样本集中，CITED4+对应的各组间的ROC曲线分析结果见图15和下表8。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.917，对应的特异性为77.8％，灵敏度为93.3％；轻度与重度相比(L/S)AUC值为0.961，对应的特异性为88.2％，灵敏度为93.3％；轻中度与重度(LM/S)相比AUC值为0.938，对应的特异性为82.9％，灵敏度为93.3％。结果表明CITED4是能够标记椎间盘髓核组织退变严重程度的生物标志物，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在极高的应用价值。In the total sample set collected by the present invention, the ROC curve analysis results of each group corresponding to CITED4+ are shown in Figure 15 and Table 8 below. Except for mild and moderate (L/M), the ROC curve AUC values of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.917, the corresponding specificity is 77.8%, and the sensitivity is 93.3%; the AUC value of mild compared with severe (L/S) is 0.961, the corresponding specificity is 88.2%, and the sensitivity is 93.3%; the AUC value of mild to moderate compared with severe (LM/S) is 0.938, the corresponding specificity is 82.9%, and the sensitivity is 93.3%. The results show that CITED4 is a biomarker that can mark the severity of degeneration of intervertebral disc nucleus pulposus tissue, especially in the diagnosis of moderate and severe, mild and severe, mild to moderate and severe degenerative tissues, and has extremely high application value.
表8 CITED4+细胞各组别ROC曲线参数
Table 8 ROC curve parameters of CITED4+ cell groups
在本发明收集得到的总的样本集中，METRN在软骨细胞的胞浆中呈阳性表达。如图16可见，随着退变严重程度增加，METRN+的阳性细胞的数量明显增多。轻度退变的METRN+细胞约1％，中度退变的METRN+细胞约占5％，而重度退变的METRN+细胞约为50％。组间统计学差异见图17，中度与重度相比(M/S)存在显著差异(P<0.001)，轻度与重度(L/S)相比存在显著差异(P<0.001)，轻中度与重度相比(LM/S)存在显著差异(P<0.001)，轻度与中重度相比(L/MS)存在显著差异(P＝0.012)。In the total sample set collected by the present invention, METRN is positively expressed in the cytoplasm of chondrocytes. As shown in Figure 16, as the severity of degeneration increases, the number of METRN+ positive cells increases significantly. Mildly degenerated METRN+ cells account for about 1%, moderately degenerated METRN+ cells account for about 5%, and severely degenerated METRN+ cells account for about 50%. The statistical differences between the groups are shown in Figure 17. There are significant differences (P<0.001) between moderate and severe (M/S), there are significant differences (P<0.001) between mild and severe (L/S), there are significant differences (P<0.001) between mild and moderate (LM/S), there are significant differences (P<0.001) between mild and moderately severe (L/MS), and there are significant differences (P=0.012).
在本发明收集得到的总的样本集中，METRN+对应的各组间的ROC曲线结果见图18和下表9。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.911，对应的特异性为77.8％，灵敏度为100％，轻度与重度相比(L/S)AUC值为0.929，对应的特异性为82.4％，灵敏度为100％，轻中度与重度(LM/S)相比AUC值为0.920，对应的特异性为80％，灵敏度为100％。结果表明METRN是能够标记椎间盘髓核组织退变严重程度的生物标志物，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在极高的应用价值。In the total sample set collected by the present invention, the ROC curve results of each group corresponding to METRN+ are shown in Figure 18 and Table 9 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.911, the corresponding specificity is 77.8%, and the sensitivity is 100%. The AUC value of mild compared with severe (L/S) is 0.929, the corresponding specificity is 82.4%, and the sensitivity is 100%. The AUC value of mild-moderate compared with severe (LM/S) is 0.920, the corresponding specificity is 80%, and the sensitivity is 100%. The results show that METRN is a biomarker that can mark the severity of degeneration of intervertebral disc nucleus pulposus tissue, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues. There is a very high application value.
表9 METRN+细胞各组别ROC曲线参数
Table 9 ROC curve parameters of each group of METRN+ cells
在本发明收集得到的总的样本集中，CITED4+METRN+对应的各组间的ROC曲线结果见图19和下表10。除轻度与中度(L/M)外，其余组别的ROC曲线AUC值均大于0.7。其中，中度与重度相比(M/S)AUC值为0.933，对应的特异性为72.2％，灵敏度为100％，轻度与重度相比(L/S)AUC值为0.953，对应的特异性为100％，灵敏度为86.7％，轻中度与重度(LM/S)相比AUC值为0.949，对应的特异性为85.7％，灵敏度为93.3％。结果表明CITED4和METRN两者联合能够标记椎间盘髓核组织退变严重程度，尤其在区别诊断中度和重度、轻度和重度、轻中度和重度退变组织中存在显著更优的应用价值。In the total sample set collected by the present invention, the ROC curve results of each group corresponding to CITED4+METRN+ are shown in Figure 19 and Table 10 below. Except for mild and moderate (L/M), the ROC curve AUC values of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.933, the corresponding specificity is 72.2%, and the sensitivity is 100%, the AUC value of mild compared with severe (L/S) is 0.953, the corresponding specificity is 100%, and the sensitivity is 86.7%, and the AUC value of mild-moderate compared with severe (LM/S) is 0.949, the corresponding specificity is 85.7%, and the sensitivity is 93.3%. The results show that the combination of CITED4 and METRN can mark the severity of degeneration of intervertebral disc nucleus pulposus tissue, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, there is a significantly better application value.
表10 CITED4+METRN+细胞各组别ROC曲线参数
Table 10 ROC curve parameters of CITED4+METRN+ cell groups
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。The above-mentioned embodiments are only used to understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.