WO2017101172A1 - 一种适用于后纵韧带骨化诊断的血清miRNA标志物及应用 - Google Patents

一种适用于后纵韧带骨化诊断的血清miRNA标志物及应用 Download PDF

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WO2017101172A1
WO2017101172A1 PCT/CN2016/000675 CN2016000675W WO2017101172A1 WO 2017101172 A1 WO2017101172 A1 WO 2017101172A1 CN 2016000675 W CN2016000675 W CN 2016000675W WO 2017101172 A1 WO2017101172 A1 WO 2017101172A1
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mirna
kit
ossification
longitudinal ligament
posterior longitudinal
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French (fr)
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刘洋
徐辰
张�浩
袁文
曹鹏
吴卉乔
陈元元
沈晓龙
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中国人民解放军第二军医大学
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Priority to US16/063,195 priority Critical patent/US11124832B2/en
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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  • the invention relates to the technical field of medical biological detection, in particular to a serum miRNAs marker and a detection application suitable for early screening and diagnosis of ossification of the posterior longitudinal ligament.
  • Ossification of the posterior longitudinal ligament is one of the common spinal disorders.
  • the ossification causes spinal cord compression, causing limbs and trunk sensation, movement and sphincter dysfunction, eventually leading to quadriplegia and bowel movements.
  • Incontinence is a disease that seriously endangers human health.
  • the OPLL Asian population is particularly common, with a Japanese incidence of 1.9 to 4.3% (Stapleton CJ, Pham MH, Attenello FJ, et al. Ossification of the posterior longitudinal ligament: genestics and pathophysiology [J]. Neurosurg Focus, 2011, 30 (3 ): E6.).
  • miRNAs are a class of non-coding short-stranded nucleotide fragments with a length of only 20-24 bp. They are highly conserved in evolution and have a wide range of roles in growth, tumorigenesis and immune response. Since the mode of action of miRNA is based on the sequence specificity of the target site, one miRNA may act on multiple mRNAs, and one mRNA may be regulated by multiple miRNAs. The deletion of individual miRNAs may not affect the development and survival of organisms.
  • the primary object of the present invention is to provide a serum miRNAs marker associated with ossification of the posterior longitudinal ligament in response to the above technical problems.
  • the first aspect of the present invention provides serum miRNAs markers suitable for early screening and diagnosis of ossification of the posterior longitudinal ligament: miRNA-563, miRNA-196b, miRNA-10a, and miRNA-129.
  • the miRNA-563 Accession number: MIMAT0003227 of the present invention, the specific sequence is as follows:
  • the miRNA-196b of the present invention Accession number: MIMAT0001080, the specific sequence is as follows:
  • the miRNA-10a of the present invention Accession number: MIMAT0000253, the specific sequence is as follows:
  • the miRNA-129 Accession number: MIMAT0000242 according to the present invention, the specific sequence is as follows:
  • the expression levels of miRNA-563, miRNA-196b, miRNA-10a and miRNA-129 in serum were positively correlated with the degree of ossification of the posterior longitudinal ligament. Therefore, by detecting the expression levels of miRNA-563, miRNA-196b, miRNA-10a, and miRNA-129 in serum, the risk and extent of ossification of the posterior longitudinal ligament in the test population can be assessed.
  • the diagnostic reagent or kit is a serological detection reagent or a detection kit.
  • the diagnostic reagent or kit detects the expression level of one or more miRNAs of miRNA-563, miRNA-196b, miRNA-10a, and miRNA-129 in the biological sample.
  • the biological sample is selected from the group consisting of fresh tissue or cells obtained from a subject, formalin-fixed or paraffin-embedded tissue or cells, blood or body fluids.
  • the biological sample is serum.
  • the diagnostic reagent or kit includes a probe, a gene chip, or a PCR primer having detection specificity for one or more of miRNA-563, miRNA-196b, miRNA-10a, and miRNA-129.
  • the diagnostic reagent or kit detects miRNA by using Real-time RT-PCR (qPCR) method An amount of reagent or kit.
  • qPCR Real-time RT-PCR
  • nucleotide sequences of the PCR primers having detection specificity for miRNA-563, miRNA-196b, miRNA-10a, and miRNA-129 are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID, respectively. NO: 7, SEQ ID NO: 8.
  • a kit for diagnosing ossification of a posterior longitudinal ligament comprising an agent for detecting a miRNA expression amount, wherein the miRNA is selected from the group consisting of miRNA-563, miRNA-196b, One or more of miRNA-10a and miRNA-129.
  • the reagent for detecting the expression level of the miRNA is a reagent for detecting the expression level of the miRNA by a Real-time RT-PCR method.
  • the kit comprises: a nucleotide sequence of a PCR primer having detection specificity for miRNA-563, miRNA-196b, miRNA-10a, miRNA-129, respectively, as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
  • a use of an agent for detecting expression levels of miRNA-563, miRNA-196b, miRNA-10a, miRNA-129 for preparing a posterior longitudinal ligament ossification diagnostic or screening kit.
  • kits for diagnosing or screening a posterior longitudinal ligament ossification according to the primers shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. Use in.
  • the kit of the present invention can screen the risk of ossification of the posterior longitudinal ligament in the population to be tested: miRNA-563, miRNA-196b, The expression level of any of miRNA-10a and miRNA-129 is higher than the normal range (the normal range of the above miRNA is 6649 ⁇ 1554, 1800 ⁇ 901, 63317 ⁇ 17258, 1075 ⁇ 651 copy number per 500 microliter per week) Venous blood serum), the risk of ossification of the posterior longitudinal ligament is higher; if the expression level of any of miRNA-563, miRNA-196b, miRNA-10a, miRNA-129 is lower than the normal range (the normal miRNA above) The range of values was 1800 ⁇ 901, 6649 ⁇ 1554, 63317 ⁇ 17258, 1075 ⁇ 651 copies per 500 microliters of peripheral venous blood serum, and the risk of ossification of the posterior longitudinal ligament was low
  • the technical scheme of the invention comprises: (1) establishing a standard standard specimen collection and processing program, that is, a standardized operation procedure (SOP); (2) establishing a system for sampling the clinical information sampling process of the test subject and a data storage and storage program; (3) Development of a Serum miRNA Screening Kit: Development of an epidemiological screening kit for OPLL based on specific serum miRNAs of the posterior longitudinal ligament ossification and healthy human controls.
  • SOP standardized operation procedure
  • the invention can be used for early diagnosis screening of ossification of the posterior longitudinal ligament in the high-risk group of the posterior longitudinal ligament.
  • the present invention detects the serum content of the above miRNA and utilizes a linear fitting formula of the four miRNAs (miRNA-563 per 500 ⁇ l peripheral venous blood serum copy number ⁇ 0.004 + miRNA-196b per 500 ⁇ l peripheral venous blood serum copy number ⁇ 0.03+miRNA-10a per 500 microliters of peripheral venous blood serum copy number ⁇ 0.003 + miRNA-129 per 500 microliters of peripheral venous blood serum copy number ⁇ 0.05-353) to obtain a fitted value, the new fitted value is greater than 0 It is more likely to have ossification of the posterior longitudinal ligament, while less than 0 may have a lower ossification of the posterior longitudinal ligament.
  • This application can be used to screen patients with posterior longitudinal ligament ossification early in order to intervene or prevent early, to delay the progress of patients and reduce the disability rate.
  • the beneficial effects of the present invention are as follows: the present invention firstly found biomarkers miRNA-563, miRNA-196b, miRNA-10a, miRNA-129 having high diagnostic value for ossification of the posterior longitudinal ligament; passing serum miRNA markers and diagnosis
  • the development and application of the kit can make the early screening of the posterior longitudinal ligament ossification more convenient and convenient, and provide a basis for the clinician to quickly and accurately grasp the patient's condition, lay a foundation for improving the clinical treatment effect, and find a new type of potential therapeutic value. Small molecule drug targets provide help.
  • Figure 1 shows the difference between miRNAs in the control group (non-posterior longitudinal ligament ossification patient tissue) and the case group (posterior longitudinal ligament ossification patient tissue) by scatter plot analysis after high-throughput miRNA sequencing. Expressed miRNA. The 10 miRNAs with the highest fold change (marked by black font) were used as candidate miRNAs for subsequent validation.
  • Figure 2 shows the serum levels of candidate miRNAs in 10 patients with stenosis of the posterior longitudinal ligament ossification and 10 patients with non-posterior longitudinal ligament ossification.
  • Figure 3 shows the detection of serum miRNA-563, miRNA-196b, miRNA-10a and miRNA-129 in 36 OPLL patients and 30 normal healthy individuals in Example 2.
  • the relative magnitude of the operating factor was determined by taking the sample with the highest CT value as a control and calculating the external component cel-miR-39 content.
  • the results showed that the expression of serum miRNAs in the OPLL group was significantly higher than that in the normal control group of the posterior longitudinal ligament (PLL).
  • PLL posterior longitudinal ligament
  • Figure 4 shows the detection of serum miRNA-563, miRNA-196b, miRNA-10a and miRNA-129 in 68 OPLL patients and 45 non-OPLL patients in Example 3.
  • the miRNA copy number (copy number/500 microliters of serum) of each sample was obtained by absolute quantification of the value of the real-time PCR and the standard curve and subjected to mapping analysis.
  • the results showed that the total expression of 4 miRNAs in the OPLL group was significantly higher than that in the control group by the method of the related reagents of the present invention. **, P ⁇ 0.01.
  • Figure 5 shows the results of the ROC curve and analysis of the results in Figure 4, showing that each of the four miRNAs has a certain specificity, and the ROC curve analysis results of the four miRNAs are significant (P ⁇ 0.05).
  • P ⁇ 0.05 Four miRNAs Logistic multivariate regression analysis was performed on the absolute quantitative values, and new fitted values representing the four miRNA levels were fitted and the fitted values were subjected to ROC test. The results showed that the specificity of the fitted values was higher than that of the individual miRNAs, and P ⁇ 0.01. The results suggest that all four miRNAs can provide important reference value for the diagnosis of OPLL. The fitting values of the four miRNAs after logistic regression can achieve higher diagnostic specificity.
  • the reagents and starting materials used in the present invention are either commercially available or can be prepared by literature methods.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions. , or in accordance with the conditions recommended by the manufacturer.
  • the preserved tissue samples are handed over to the company (High-flux sequencing company such as Lie Bing Information and Bohao Bio) for high-throughput RNA extraction.
  • the company High-flux sequencing company such as Lie Bing Information and Bohao Bio
  • the samples passed by the quality inspection are constructed and sequenced by the company, and the sequencing is performed by using Hiseq2000 (illumina) or above.
  • miRNAs with significant differences in expression between the case group and the control group were screened, and 10 miRNAs with the highest FDR ⁇ 0.5 and the highest fold change were selected for subsequent validation studies.
  • the case group was collected from 36 cases in Shanghai Changzheng Hospital from June 2015 to September 2015. All patients were diagnosed with posterior longitudinal ligament ossification by cervical or thoracic CT three-dimensional reconstruction. No treatment was performed before blood collection.
  • the control group was 30 healthy individuals who were screened for community disease during the same period. All samples were informed of the purpose of collection and consent of the patients before collection. There was no significant difference in the age, smoking, drinking and other factors between the case group and the control group (P>0.05).
  • Takara RNAiso (provided by Takara) or TRIzol Reagent (provided by Life Technology, or equivalent phenol chloroform RNA extraction reagent) to 500 ⁇ l of serum sample to a total volume of 1.2 ml, and place at room temperature (about 25 ° C) for 10 min.
  • PCR product content was calculated by relative Ct value method for relative quantification.
  • the comparison of the Ct value method assumes that the number of products is doubled per cycle, and the Ct value is obtained in the exponential phase of the PCR reaction to reflect the amount of the starting template.
  • Example 3 OPLL screening of patients by detecting serum OPLL-specific miRNAs
  • the case group was 68 cases of OPLL collected from the Department of Spinal Surgery, Shanghai Changzheng Hospital from June 2015 to October 2015. All patients were diagnosed with posterior longitudinal ligament ossification by cervical CT three-dimensional reconstruction. No treatment was performed before blood collection.
  • the control group received 45 patients who were admitted to the orthopedic clinic. None of the 45 patients had obvious OPLL symptoms, signs or imaging findings. All samples were informed of the purpose of the collection prior to collection and patient consent was obtained. There was no significant difference in the age, smoking, drinking and other factors between the case group and the control group (P>0.05).

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Abstract

一组适用于后纵韧带骨化症早期筛查及诊断的血清miRNA生物标志物miRNA-563、miRNA-196b、miRNA-10a、miRNA-129，该标志物可用于制备后纵韧带骨化的诊断试剂或试剂盒。通过血清miRNA标志物和诊断试剂盒的研制和应用，可以使后纵韧带骨化的早期筛查更加方便易行，为临床医生快速并准确掌握患者病情，为提高临床治疗效果奠定基础，并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。

Description

一种适用于后纵韧带骨化诊断的血清miRNA标志物及应用 技术领域
本发明涉及医学生物检测技术领域，具体地说，是一种适用于后纵韧带骨化症早期筛查及诊断的血清miRNAs标志物及检测应用。
背景技术
后纵韧带骨化症(ossification of the posterior longitudinal ligament，OPLL)是常见的脊柱疾患之一，骨化物造成脊髓压迫，引起患者四肢与躯干感觉、运动及括约肌功能障碍，最终导致四肢瘫痪和大小便失禁，是严重危害人类健康的疾病。OPLL亚洲人群尤其多见，日本发病率为1.9～4.3％(Stapleton CJ，Pham MH，Attenello FJ，et al.Ossification of the posterior longitudinal ligament：genetics and pathophysiology[J].Neurosurg Focus，2011，30(3)：E6.)。由于骨化物质地坚硬，减压手术风险大，容易因脊髓损伤导致患者发生瘫痪等严重并发症。在诊断方面，由于其早期首发症状往往来源于脊髓神经根受压，与其他脊柱脊髓疾患易混淆造成漏诊误诊；而从治疗角度考虑，由于骨化范围广，脊髓压迫重，椎管的储备间隙小，其手术治疗是脊柱外科的巨大挑战(Seichi A，Hoshino Y，Kimura A，et al.Neurological complications of cervical laminoplasty for patients with ossification of the posterior longitudinal ligament-a multi-institutional retrospective study.Spine(Phila Pa 1976).2011，36(15)：E998-1003.)。因此，为后纵韧带骨化的早期诊断寻找高敏感性或特异性的早期筛查生物标志物已成为一个急需解决的问题。
近年来，随着人类基因组计划的进一步开展，大量的非编码序列越来越受到关注，其中microRNA(miRNA)的发现最引人注目。miRNA是一类全长仅有20-24bp的非编码短链核苷酸片段，进化上具有高度的保守性，其在生长发育、肿瘤发生、免疫应答等方面具有广泛的作用。由于miRNA作用方式的是以靶位点序列特异性为基础，一个miRNA可能作用于多个mRNA，而一个mRNA又可能接受多个miRNA的调控，个别miRNA的缺失可能对生物的发育及生存并没有明显的影响，但却为某些疾病的发生埋下了伏笔(Flynt AS，Lai EC.Biological principles of microRNA-mediated regulation：shared themes amid diversity.Nat Rev Genet.2008，9(11)：831-842.)。而由于miRNA作用方式的特殊性，使其在许多慢性疾病中都起到了至关重要的作用(Ceribelli A，Nahid MA，Satoh M，et al.MicroRNAs in rheumatoid arthritis.FEBS Lett.2011，585(23)：3667-74.)，其有望成为重大疾病发生、诊断和预后的重要候选标志物。现已证明miRNA在外周血中性质稳定且存在显著的疾病特异性， 但含量较低、检测不易。因此开发易于检测、成本低廉、快速有效的检测试剂盒成品则能够有效提高检测效率并达到良好的可重复性。然而，目前还没有应用血清miRNA标志物对后纵韧带骨化早期筛查的研究报道，若能筛选出后纵韧带骨化异常表达的血清miRNA作为生物标志物，并研制相应的筛选试剂盒，必将对我国后纵韧带骨化的早期诊断具有重要意义。
发明内容
本发明的首要目的是针对上述技术问题，提出一种与后纵韧带骨化相关的血清miRNAs标志物。
为解决上述技术问题，本发明的第一方面，提供适用于后纵韧带骨化症早期筛查及诊断的血清miRNAs标志物：包括miRNA-563、miRNA-196b、miRNA-10a、miRNA-129。
本发明所述的miRNA-563，Accession number：MIMAT0003227，具体序列如下：
agguugacauacguuuccc(SEQ ID NO：1)。
本发明所述的miRNA-196b，Accession number：MIMAT0001080，具体序列如下：
uagguaguuuccuguuguuggg(SEQ ID NO：2)。
本发明所述的miRNA-10a，Accession number：MIMAT0000253，具体序列如下：
uacccuguagauccgaauuugug(SEQ ID NO：3)。
本发明所述的miRNA-129，Accession number：MIMAT0000242，具体序列如下：
cuuuuugcggucugggcuugc(SEQ ID NO：4)。
miRNA-563、miRNA-196b、miRNA-10a、miRNA-129在血清中的表达水平与后纵韧带骨化程度成正相关。因此，通过检测血清中miRNA-563、miRNA-196b、miRNA-10a、miRNA-129的表达水平，可以评估待检人群中后纵韧带骨化的风险及程度。
本发明的第二方面，提供上述血清miRNA标志物在制备后纵韧带骨化的诊断试剂或试剂盒中的应用。优选的，所述的诊断试剂或试剂盒为血清学检测试剂或检测试剂盒。
所述的诊断试剂或试剂盒检测生物样品中miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上的miRNA的表达量。所述的生物样品选自：获自对象的新鲜组织或细胞、福尔马林固定或石蜡包埋组织或细胞、血液或体液。优选的，所述的生物样品为血清。
所述的诊断试剂或试剂盒包括：对miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上具有检测特异性的探针、基因芯片，或PCR引物。
优选的，所述的诊断试剂或试剂盒为采用Real-time RT-PCR(qPCR)方法检测miRNA 表达量的试剂或试剂盒。
优选的，所述的对miRNA-563、miRNA-196b、miRNA-10a、miRNA-129具有检测特异性的PCR引物的核苷酸序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8所示。
本发明的第三方面，提供一种诊断后纵韧带骨化的试剂盒，所述的试剂盒中包括用于检测miRNA表达量的试剂，所述的miRNA选自miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上。
优选的，所述的用于检测miRNA表达量的试剂为采用Real-time RT-PCR方法检测miRNA表达量的试剂。
优选的，所述的试剂盒中包括：对miRNA-563、miRNA-196b、miRNA-10a、miRNA-129具有检测特异性的PCR引物的核苷酸序列，分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8所示。
本发明的第四方面，提供检测miRNA-563、miRNA-196b、miRNA-10a、miRNA-129表达水平的试剂在制备后纵韧带骨化诊断或筛查用试剂盒中的用途。
本发明的第五方面，提供如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示的引物在制备后纵韧带骨化诊断或筛查用试剂盒中的用途。
本发明的试剂盒通过检测miRNA-563、miRNA-196b、miRNA-10a、miRNA-129的表达水平，可以筛查待检测人群患后纵韧带骨化的风险：若miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中任一miRNA的表达水平高于正常值范围(上述miRNA的正常值范围依次为6649±1554，1800±901，63717±17258，1075±651拷贝数每500微升外周静脉血血清)，则患后纵韧带骨化的风险较高；若miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中任一miRNA的表达水平低于正常值范围(上述miRNA的正常值范围依次为1800±901，6649±1554，63717±17258，1075±651拷贝数每500微升外周静脉血血清)，则患后纵韧带骨化的风险低。
本发明的技术方案包括：(1)建立统一标准的标本采集及处理程序，即标准化操作程序(SOP)；(2)建立系统的待测者临床信息取样流程及资料保管储存程序；(3)血清miRNA筛查试剂盒的研制：根据后纵韧带骨化和健康人对照的特异血清miRNA开发miRNA对于OPLL的流行病学筛查试剂盒。
本发明可用于对后纵韧带骨化高危人群进行后纵韧带骨化症疾病的早期诊断筛查，本发明通过检测上述miRNA的血清含量同时利用4种miRNA的线性拟合公式(miRNA-563每500微升外周静脉血血清拷贝数×0.004+miRNA-196b每500微升外周静脉血血清拷贝数 ×0.03+miRNA-10a每500微升外周静脉血血清拷贝数×0.003+miRNA-129每500微升外周静脉血血清拷贝数×0.05-353)得出拟合值，新的拟合值大于0则具有较高可能患后纵韧带骨化症，而小于0则患后纵韧带骨化症的可能较低。此应用可早期筛查后纵韧带骨化症的患者，以期及早进行干预或预防，最大限度的延缓患者病情进展，降低致残率。
本发明的有益效果在于：本发明首次发现了对后纵韧带骨化具有较高诊断价值的生物标记物miRNA-563、miRNA-196b、miRNA-10a、miRNA-129；通过血清miRNA标志物和诊断试剂盒的研制和应用，可以使后纵韧带骨化的早期筛查更加方便易行，为临床医生快速并准确掌握患者病情，为提高临床治疗效果奠定基础，并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。
附图说明
图1示高通量miRNA测序后通过将对照组(非后纵韧带骨化症患者组织)与病例组(后纵韧带骨化症患者组织)的miRNA平均值进行散点图分析后，显示差异表达的miRNA。其中变化倍数最高的10个miRNA(由黑色字体标出)被作为进行后续验证的候选miRNA。
图2为选取10例后纵韧带骨化症患者血清以及10例非后纵韧带骨化症患者血清进行候选miRNA的血清表达量检测。通过real-time PCR法，将测得的miRNA表达量进行统计分析后我们可以看到骨化症患者血清中miRNA-563、miRNA-196b、miRNA-10a及miRNA-129的表达量明显高于对照组，提示该4个miRNA可能具有诊断价值，值得进一步研究。**，P＜0.01。
图3为实施例2中36个OPLL患者及30个正常健康人的血清miRNA-563、miRNA-196b、miRNA-10a及miRNA-129的检测情况。通过将CT值最高的样本作为对照，并通过计算外参cel-miR-39含量排除操作因素得出的相对量值。结果显示利用本发明相关试剂方法检测OPLL组患者血清miRNAs表达量均显著高于后纵韧带(PLL)正常对照组。*，P＜0.05；**，P＜0.01。
图4为实施例3中68个OPLL患者及45个非OPLL患者的血清miRNA-563、miRNA-196b、miRNA-10a及miRNA-129的检测情况。通过将real-time PCR的值与标准曲线进行绝对定量，得出每个样本的miRNA拷贝数(拷贝数/500微升血清)并进行作图分析。结果显示利用本发明相关试剂方法检测OPLL组患者血清4个miRNAs总体表达量均显著高于对照组。**，P＜0.01。
图5将图4中的结果进行ROC曲线的作图及分析，可见4个miRNA每一个单独都具有一定的特异性，且4种miRNA的ROC曲线分析结果均较为显著(P＜0.05)。将4种miRNA 的绝对定量值进行logistic多因素回归分析，拟合出新的代表4种miRNA水平的拟合值，将该拟合值进行ROC检验。结果显示拟合值的特异性较单个miRNA特异性高，且P＜0.01。结果提示4种miRNA均能对诊断OPLL提供重要参考价值，其中将4种miRNA进行logistic回归后的拟合值能够达到更高的诊断特异性。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等《分子克隆：实验室指南》(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照常规条件，或按照制造厂商所建议的条件。
实施例1
一、研究对象
选取后纵韧带骨化症患者的后纵韧带组织(n＝3)以及外伤手术中正常后纵韧带组织(n＝3)进行高通量测序及miRNA组的生物信息学分析。病例组与对照组的年龄、吸烟、饮酒等因素的分部差异无统计学意义(P＞0.05)。
二、研究方法
1.手术中选取患者的韧带组织，立即转入RNA later(Ambion公司或其它同等产品)低温短暂保存。转运至实验室后立即进行-80℃低温冻存。
2.待样本收齐后将保存好的组织样品一并交予公司(烈冰信息、伯豪生物等高通量测序公司)进行高通量RNA的抽提及质检。
3.质检通过的样品由公司进行miRNA库的构建及测序，测序采用Hiseq2000(illumina公司)或以上平台。
4.测序数据进行均一化处理后，对表达量在病例组及对照组间存在明显差异的miRNA进行筛选，选取FDR＜0.5且变化倍数最高的10个miRNA进行后续验证研究。
5.将候选miRNA在额外的6个后纵韧带骨化症组织以及正常韧带组织中进行表达量的验证。
6.为了筛选出在外周血具有诊断意义的miRNA，抽取10例后纵韧带骨化症患者以及10例正常人静脉血5ml，并于室温凝血后作500×g短暂离心3min，取上清500微升，利用QIAGEN血清miRNA回收试剂盒进行miRNA的收集、逆转录及检测。
三、实验结果
通过高通量测序及生物信息学分析对后纵韧带骨化症患者的后纵韧带组织以及外伤手术中正常后纵韧带中的miRNA进行高通量测序筛选，选取表达差异最高的10条miRNA作为研究对象，如图1所示。候选miRNA在经过组织层面表达验证后遂进行后纵韧带骨化症患者及正常患者的静脉血血清miRNA的检测。如图2所示，上述10条差异miRNA在后纵韧带骨化症患者血清中仅有miRNA-563、miRNA-196b、miRNA-10a及miRNA-129相较于正常人血清中含量明显升高，提示miRNA-563、miRNA-196b、miRNA-10a及miRNA-129可能具有后纵韧带骨化症的血清学特异性，可进行进一步的深入研究。
实施例2：针对入院患者进行血清OPLL特异性miRNA筛查
一、研究对象
病例组为2015年6月至2015年9月在上海长征医院收集的36例病例，均经颈椎或胸椎CT三维重建确诊后纵韧带骨化症，采血前未经任何治疗。对照组为同期进行社区疾病筛查的健康个体30例，所有样本采集前告知采集目的并获得患者同意。病例组与对照组的年龄、吸烟、饮酒等因素的分部差异无统计学意义(P＞0.05)。
二、研究方法
1.晨起空腹，使用EDTA抗凝管采肘静脉血5ml，采血后轻轻摇动，使抗凝剂与血液混合均匀，室温静置40min之后1800转离心20min，将血清与血细胞分离，装入1.5ml EP管中，-80℃保存；
2.将Takara RNAiso(Takara公司提供)或TRIzol Reagent(Life Technology提供，或同等酚氯仿RNA抽提试剂)加入至500μl血清样本至总体积1.2ml，并于室温(25℃左右)下放置10min，确保完全裂解；加入氯仿(0.2ml氯仿/每1ml RNA抽提试剂)剧烈上下震荡15s后，在室温下静置5min以上待其分层，上层水相即为所需RNA；13000×g低温离心15min(4℃)，将上层澄清水相用无核酶枪头小心地吸出700μl，加入至等量异丙醇中，充分颠倒混匀于室温下静置约15min以上。再次13000×g，低温离心10min(4℃)。小心保留沉淀并弃去上清，加入75％(V/V)乙醇(1ml 75％乙醇/ml裂解液)，颠倒混合样本；7500×g低温离心5min(4℃)。小心弃去上清，同时于空气中干燥5min(注意不可使RNA过分干燥，或将影响其溶解)；加入14μl预热的DEPC水溶解总RNA，同时加入1μl人工合成的线虫miRNA cel-miR-39(5’-UCACCGGGUGUAAAUCAGCUUG-3’，10nM，SEQ ID NO：10)作为外参直接加入每个样品，至于4℃冰箱用于逆转录。
3.Real-time RT-PCR(qPCR)方法
(1)取受试者血清总RNA，利用TOYOBO ReverTra逆转录试剂盒(或同等产品)制备20μl体积的逆转录反应体系，并通过37℃水浴锅(或同等环境下)进行逆转录15min获 得cDNA样品；
(2)引物设计，逆转录所用引物如下；
miR-563逆转录引物：
Figure PCTCN2016000675-appb-000001
miR-196b逆转录引物：
Figure PCTCN2016000675-appb-000002
miR-10a逆转录引物：
Figure PCTCN2016000675-appb-000003
miR-129逆转录引物：
Figure PCTCN2016000675-appb-000004
Cel-39逆转录引物：
Figure PCTCN2016000675-appb-000005
(3)加入
Figure PCTCN2016000675-appb-000006
 Green Realtime PCR Master Mix进行PCR反应，反应体系如下：
Figure PCTCN2016000675-appb-000007
PCR反应步骤：
Figure PCTCN2016000675-appb-000008
(4)检测并比较后纵韧带骨化病例与健康人对照血清样本中miRNA的含量的变化。PCR产物含量计算采用比较Ct值法进行相对定量。比较Ct值法前提是假设每个循环增加一倍的产物数量，在PCR反应的指数期得到Ct值来反应起始模板的量，一个循环(Ct＝1)的不同相当于起始模板数2倍的差异。
定义：ΔCt＝Ct目的基因-Ct内标
ΔΔCt＝(Ct目的基因-Ct内标)已处理-(Ct目的基因-Ct内标)未处理
RQ＝2-ΔΔCt
利用EXCEL里面的统计分析工具，计算各组的平均值和标准差，两组之间用t检验，P＜0.05认为有统计学意义，P＜0.01认为差异显著。
三、研究结果
病例组血清miRNA-563、miRNA-196b、miRNA-10a、miRNA-129表达水平较对照组显著上调，具体如表1和图3所示：
表1：对照组与病例组血清miRNA水平比较(ΔCT)(X±SD)
Figure PCTCN2016000675-appb-000009
上述结果显示血清miRNA-563、miRNA-196b、miRNA-10a、miRNA-129水平在对照组及病例组中具有明显差异，提示其中任一miRNA的上调均对发生韧带骨化具有提示意义。
实施例3：通过检测血清OPLL特异性miRNA对患者进行OPLL的筛查
一、研究对象
病例组为2015年6月至2015年10月在上海长征医院脊柱一科收集的68例OPLL病例，均经颈椎CT三维重建确诊后纵韧带骨化症，采血前未经任何治疗。对照组为骨科门诊接诊病人45例，该45例患者均未见明显OPLL症状、体征或影像学表现。所有样本采集前告知采集目的并获得患者同意。病例组与对照组的年龄、吸烟、饮酒等因素的分部差异无统计学意义(P＞0.05)。
二、研究方法
同实施例2。
为了明确血清中4种miRNA的具体含量，我们利用标准曲线及绝对定量real-time PCR对上述4种指标进行分析。
此外，为了检测前述4种miRNA指标的特异性及敏感性，利用SPSS软件对测得数值进行统计分析，并通过线性回归方程拟合联合参数，将4种miRNA拟合成新值，并对该新值进行特异性的检验。
三、研究结果
病例组血清miRNA-563、miRNA-196b、miRNA-10a、miRNA-129表达水平较对照组显著上调，具体如图4所示。
利用SPSS进行ROC曲线分析，我们发现上述4种miRNA均有较好的特异性及敏感性，如图5所示。
为了将4种阳性指标联合分析，我们采取线性回归方程进行拟合，拟合出的新值其ROC曲线分析结果显示其特异性及敏感性均较4种miRNA单一作为诊断指标要更高，如图5所示。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (10)

  1. miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，所述的miRNA选自miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上，所述的miRNA-563、miRNA-196b、miRNA-10a、miRNA-129的核苷酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示。
  2. 根据权利要求1所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的诊断试剂或试剂盒为血清学诊断试剂或诊断试剂盒。
  3. 根据权利要求1所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的诊断试剂或试剂盒检测生物样品中miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上的miRNA的表达量。
  4. 根据权利要求3所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的生物样品选自：获自对象的新鲜组织或细胞、福尔马林固定或石蜡包埋组织或细胞、血液或体液。
  5. 根据权利要求3所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的生物样品为血清。
  6. 根据权利要求1所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的诊断试剂或试剂盒包括：对miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上具有检测特异性的探针、基因芯片，或PCR引物。
  7. 根据权利要求1所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的诊断试剂或试剂盒为采用Real-time RT-PCR方法检测miRNA表达量的试剂或试剂盒。
  8. 根据权利要求6所述的miRNA在制备后纵韧带骨化的诊断试剂或试剂盒中的应用，其特征在于，所述的对miRNA-563、miRNA-196b、miRNA-10a、miRNA-129具有检测特异性的PCR引物的核苷酸序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8所示。
  9. 一种诊断后纵韧带骨化的试剂盒，其特征在于，所述的试剂盒中包括用于检测miRNA表达量的试剂，所述的miRNA选自miRNA-563、miRNA-196b、miRNA-10a、miRNA-129中的一种或两种以上。
  10. 根据权利要求9所述的诊断后纵韧带骨化的试剂盒，其特征在于，所述的用于检测miRNA表达量的试剂为采用Real-time RT-PCR方法检测miRNA表达量的试剂。
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