CN114574573A - 测定尿液微囊泡中rna含量的试剂在制备糖尿病肾病诊断试剂中的应用 - Google Patents
测定尿液微囊泡中rna含量的试剂在制备糖尿病肾病诊断试剂中的应用 Download PDFInfo
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Abstract
本发明公开了测定尿液微囊泡中RNA含量的试剂在制备糖尿病肾病诊断试剂中的应用。与糖尿病肾病相关的尿液微囊泡RNA标志物，选自下列环状RNA中的任意一种或多种：MS4A6A、CPA3、CXCL6和TAC1。定量检测所述的尿液微囊泡RNA标志物的试剂在制备糖尿病肾病诊断试剂或辅助诊断试剂中的应用。发明人通过高通量测序和定量PCR发现尿液微囊泡中4种RNA在糖尿病肾病患者中明显上升，并且能够有效的将糖尿病肾病患者和非糖尿病肾病患者区分开，ROC曲线下AUC均大于0.85，具备诊断价值。总体来说，这4个优选的尿液微囊泡RNA可作为检测标志物应用于糖尿病肾病的诊断。
Description
技术领域
本发明属于医学诊断领域，涉及尿液微囊泡中RNA在糖尿病肾病诊断中的应用。
背景技术
糖尿病肾病(diabetic nephropathy,DN)是糖尿病最主要的并发症之一,约有30％的1型糖尿病患者和约40％的2型糖尿病患者会出现糖尿病肾病。糖尿病肾病不仅是导致终末期肾脏病的首位病因，而且也是糖尿病患者死亡的主要因素之一。早期糖尿病肾病存在一定的可逆性，若能早期快速准确的诊断并给予正确、有效的治疗措施就能够阻止患者肾脏的进一步恶化，避免肾衰竭的发生。然而囿于糖尿病肾病早期无明显症状，在日常极容易被忽略。糖尿病肾病患者被诊断发现时，往往已经处于不可逆状态，只能进行有限的延缓性治疗，肾脏会不断随着时间逐渐衰竭，直至完全丧失功能。目前糖尿病肾病的诊断主要依据尿微量白蛋白，但白蛋白缺乏特异性，通常需要肾活检来进一步确诊。肾活检为有创检查，临床上存在并发出血、感染等风险而不易被患者接受，所以寻找一种安全且具有早期诊断价值的生物标志物对糖尿病肾病的早期诊断至关重要。
微囊泡是一种由活细胞释放的具膜囊泡，其中包含大量的蛋白质、DNA和RNA。机体细胞在不同的生理和病理状态下会释放不同的微囊泡，并且微囊泡中包含的蛋白质、DNA和RNA的种类和含量也会发生相应的变化。因此，微囊泡中的蛋白质、DNA和RNA种类和含量能够反应机体细胞所处的生理和病理状态，可以作为疾病诊断的标志物。微囊泡被证实广泛分布于尿液、唾液、血液、羊水、脑脊液等各种体液中。相比于其它体液，尿液具有取样安全简单、样本量大且无创等优点，因此，尿液微囊泡是泌尿系统疾病的诊断标志物的理想来源。
发明内容
本发明的目的是提供与糖尿病肾病相关的尿液微囊泡RNA标志物。
本发明的另一目的是提供所述标志物的应用。
本发明的又一目的是提供检测所述标志物的试剂及其应用。
本发明的目的可通过以下技术方案实现：
与糖尿病肾病相关的尿液微囊泡RNA标志物，选自下列环状RNA中的任意一种或多种：MS4A6A、CPA3、CXCL6和TAC1。
本发明所述的尿液微囊泡RNA标志物在制备糖尿病肾病诊断试剂中的应用。
定量检测所述的尿液微囊泡RNA标志物的试剂在制备糖尿病肾病诊断试剂或辅助诊断试剂中的应用。
作为本发明的一种优选，定量检测所述的尿液微囊泡RNA标志物的试剂为检测所述尿液微囊泡RNA标志物的引物、探针或基因芯片。
作为本发明的进一步优选，检测MS4A6A的引物如SEQ ID NO.1和SEQ ID NO.2所示；检测CPA3的引物如SEQ ID NO.3和SEQ ID NO.4所示；检测CXCL6的引物如SEQ ID NO.5和SEQ ID NO.6所示；检测TAC1的引物如SEQ ID NO.7和SEQ ID NO.8所示。
一种糖尿病肾病诊断或辅助诊断试剂盒，其特征在于包含定量检测MS4A6A、CPA3、CXCL6和TAC1中的任意两种或两种以上的试剂。
作为本发明的一种优选，所述的定量检测MS4A6A、CPA3、CXCL6和TAC1中的任意两种或两种以上的试剂为检测所述RNA的引物、探针或基因芯片。
8、根据权利要求7所述的糖尿病肾病诊断或辅助诊断试剂盒，其特征在于检测MS4A6A的引物如SEQ ID NO.1和SEQ ID NO.2所示；检测CPA3的引物如SEQ ID NO.3和SEQID NO.4所示；检测CXCL6的引物如SEQ ID NO.5和SEQ ID NO.6所示；检测TAC1的引物如SEQID NO.7和SEQ ID NO.8所示。
有益效果：
尿液微囊泡表面富含糖基化修饰的蛋白，这些糖基化修饰的蛋白能够与偶联在磁珠上的凝集素结合，从而通过磁性分离的方法就可以将尿液中的微囊泡分离出来。本发明利用凝集素偶联的磁珠捕获尿液中的微囊泡，通过高通量测序和定量PCR技术鉴别糖尿病肾病与糖尿病非肾病(糖尿病患者，且10年以上肾脏无异常)以及其它类型慢性肾病尿液中微囊泡包含的核酸种类与含量的区别。结果发现，尿液微囊泡中的MS4A6A、CPA3、CXCL6和TAC1四个基因的mRNA含量在糖尿病肾病患者尿液微囊泡中显著上升，并可以作为糖尿病肾病诊断的标志物。
发明人通过高通量测序和定量PCR发现尿液微囊泡中4种RNA在糖尿病肾病患者中明显上升，不仅能够有效的将糖尿病肾病患者和糖尿病非肾病(糖尿病患者，且10年以上肾脏无异常)患者区分开，其准确率和精确率分别位93.75和95.71；而且还能够将糖尿病肾病患者与其它类型慢性肾病患者区分开，其准确率和精确率分别位91.39和90.13。总体来说，这4个优选的尿液微囊泡RNA可作为检测标志物应用于糖尿病肾病的诊断。
附图说明
图1显示了通过高通量测序检测利用凝集素偶联的磁珠富集的尿液微囊泡内的RNA表达谱。(A)热图。其中红色表示上升的RNA，蓝色表示下降的RNA。(B)tSNE分析图。由tSNE分析表明，尿液微囊泡内的RNA可以将糖尿病肾病患者与非糖尿病肾病的糖尿病患者区分成两群。(C)火山图。绿色的点代表在糖尿病肾病患者尿液微囊泡内下降的RNA；红色的点代表在糖尿病肾病患者尿液微囊泡内上升的RNA；蓝色的点代表在糖尿病肾病患者与非糖尿病肾病的糖尿病患者尿液微囊泡内不变的RNA。DN：糖尿病肾病；diabetes：非糖尿病肾病的糖尿病患者。
图2显示了MS4A6A(A)、CPA3(B)、CXCL6(C)和TAC1(D)在来自糖尿病肾病(DN)、非糖尿病肾病的其它肾病患者(CKD(NO-DN))和非糖尿病肾病的糖尿病患者(diabetes)尿液微囊泡内的含量。
图3.显示了MS4A6A(A)、CPA3(B)、CXCL6(C)和TAC1(D)鉴别糖尿病肾病(DN)与非糖尿病肾病的糖尿病患者(diabetes)的ROC曲线。
图4.显示了MS4A6A、CPA3、CXCL6和TAC1两两联合鉴别糖尿病肾病(DN)与非糖尿病肾病的糖尿病患者(diabetes)的ROC曲线。
图5.显示了MS4A6A、CPA3、CXCL6和TAC1三三联合鉴别糖尿病肾病(DN)与非糖尿病肾病的糖尿病患者(diabetes)的ROC曲线。
图6.显示了MS4A6A、CPA3、CXCL6和TAC1四个指标联合鉴别糖尿病肾病(DN)与非糖尿病肾病的糖尿病患者(diabetes)的ROC曲线。
图7.显示了MS4A6A(A)、CPA3(B)、CXCL6(C)和TAC1(D)鉴别糖尿病肾病(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))的ROC曲线。
图8.显示了MS4A6A、CPA3、CXCL6和TAC1两两联合鉴别糖尿病肾病(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))的ROC曲线。
图9.显示了MS4A6A、CPA3、CXCL6和TAC1三三联合鉴别糖尿病肾病(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))的ROC曲线。
图10.显示了MS4A6A、CPA3、CXCL6和TAC1四个指标联合鉴别糖尿病肾病(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))的ROC曲线。
具体实施方式
实施例1尿液微囊泡中RNA表达谱鉴定
收集9例糖尿病肾病患者和13例非糖尿病肾病患者尿液各10ml，分离尿液中微囊泡，利用高通量测序检测尿液微囊泡中RNA表达。
1)来自每个检测对象的10ml尿液，在转速3000g、室温的条件下离心30分钟，去除沉淀保留上清；
2)将上一步获得的上清在转速10000g、室温的条件下离心30分钟，去除沉淀保留上清；
3)向上一步获得的上清中加入1ml凝集素(lectin)偶联的磁珠,混合均匀后4度孵育4h，随后利用磁性分离柱分离磁珠；
4)收集磁珠，利用洗脱缓冲液(0.1M Glycine，0.1％(v/v)Tween-20，pH2.5)将磁珠结合的尿液微囊泡洗脱下来，利用Trizol提取外泌体内RNA，通过高通量测序，检测尿液微囊泡内RNA。
检测结果
对分离的尿液微囊泡提取RNA后，利用高通量测序检测尿液微囊泡内RNA。检测结果发现糖尿病肾病患者与非糖尿病肾病的糖尿病患者尿液微囊泡内都存在这丰富的RNA，并且热图(图1A)，tSNE分析图(图1B)和火山图(图1C)分析表明糖尿病肾病患者与非糖尿病肾病的糖尿病患者相比，尿液微囊泡内RNA存在显著差异。进一步分析数据发现MS4A6A、CPA3、CXCL6和TAC1这4条RNA具有显著性差异。
实施例2尿液微囊泡中能够作为糖尿病肾病诊断的RNA定量检测
收集100例糖尿病肾病患者(DN)、50例非糖尿病肾病的糖尿病患者(diabetes)，50例非糖尿病肾病的其它肾病患者(CKD(NO-DN))尿液各5ml，分离尿液微囊泡并利用定量PCR检测尿液微囊泡内RNA的表达。
1)来自每个检测对象的5ml尿液，在转速3000g、室温的条件下离心30分钟，去除沉淀保留上清；
2)将上一步获得的上清在转速10000g、室温的条件下离心30分钟，去除沉淀保留上清；
3)向上一步获得的上清中加入0.5ml凝集素(lectin)偶联的磁珠,混合均匀后4度孵育4h，随后利用磁性分离柱分离磁珠；
4)收集磁珠，利用洗脱缓冲液(0.1M Glycine，0.1％(v/v)Tween-20，pH2.5)将磁珠结合的尿液微囊泡洗脱下来，利用Trizol提取外泌体内RNA，通过定量PCR，利用RNA特异性引物(表1)检测尿液微囊泡内MS4A6A、CPA3、CXCL6和TAC1四种RNA。表2引物序列
检测结果
对分离的尿液微囊泡提取RNA后，利用RNA特异性引物对RNA进行定量PCR(qRT-PCR)检测，检测结果表明MS4A6A、CPA3、CXCL6和TAC1含量在糖尿病肾病患者尿液微囊泡中显著高表达(图2)。
利用受试者工作特征曲线(receiver operating characteristic curve,简称ROC曲线)分析MS4A6A、CPA3、CXCL6和TAC1在区分糖尿病肾病(DN)和非糖尿病肾病的糖尿病患者(diabetes)时发现：当MS4A6A、CPA3、CXCL6和TAC1单独使用时，其曲线下面积分别为：0.8571,0.8016,0.8730和0.7857(图3A-D)；通过二元逻辑回归分析，当MS4A6A、CPA3、CXCL6和TAC1两两组合用于区分糖尿病肾病患者(DN)与非糖尿病肾病的糖尿病患者(diabetes)时ROC曲线下面积为：0.817，0.913，0.841，0.913，0.913，0.865(图4A-F)；当MS4A6A、CPA3、CXCL6和TAC1三三组合用于区分糖尿病肾病患者(DN)与非糖尿病肾病的糖尿病患者(diabetes)时ROC曲线下面积为0.929，0.913，0.921，0.944(图5A-D)；当MS4A6A、CPA3、CXCL6和TAC1四个指标联合使用用于区分糖尿病肾病患者(DN)与非糖尿病肾病的糖尿病患者(diabetes)时ROC曲线下面积为0.952(图6)。
利用受试者工作特征曲线(receiver operating characteristic curve,简称ROC曲线)分析MS4A6A、CPA3、CXCL6和TAC1在区分糖尿病肾病(DN)和非糖尿病肾病的其它慢性肾病患者(CKD(NO-DN))时发现：当MS4A6A、CPA3、CXCL6和TAC1单独使用时，其曲线下面积分别为：0.7360,0.8478,0.8075和0.7764(图7A-D)；通过二元逻辑回归分析，当MS4A6A、CPA3、CXCL6和TAC1两两组合用于糖尿病肾病患者(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))时ROC曲线下面积为：0.8494，0.8075，0.7764，0.8494，0.8494，0.7764(图8A-F)；当MS4A6A、CPA3、CXCL6和TAC1三三组合用于区分糖尿病肾病患者(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))时ROC曲线下面积为0.8494，0.8494，0.7764，0.8494(图9A-D)；当MS4A6A、CPA3、CXCL6和TAC1四个指标联合使用用于区分糖尿病肾病患者(DN)与非糖尿病肾病的其它肾病患者(CKD(NO-DN))时ROC曲线下面积为0.9161(图10)。
序列表
<110> 南京羿检医学科技有限公司
中国药科大学
<120> 测定尿液微囊泡中RNA含量的试剂在制备糖尿病肾病诊断试剂中的应用
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Claims (8)
1.与糖尿病肾病相关的尿液微囊泡RNA标志物，其特征在于选自下列环状RNA中的任意一种或多种：MS4A6A、CPA3、CXCL6和TAC1。
2.权利要求1所述的尿液微囊泡RNA标志物在制备糖尿病肾病诊断试剂中的应用。
3.定量检测权利要求1所述的尿液微囊泡RNA标志物的试剂在制备糖尿病肾病诊断试剂或辅助诊断试剂中的应用。
4.根据权利要求3所述的应用，其特征在于所述的定量检测权利要求1所述的尿液微囊泡RNA标志物的试剂为检测所述尿液微囊泡RNA标志物的引物、探针或基因芯片。
5.根据权利要求4所述的应用，其特征在于检测MS4A6A的引物如SEQ ID NO.1和SEQ IDNO.2所示；检测CPA3的引物如SEQ ID NO.3和SEQ ID NO.4所示；检测CXCL6的引物如SEQ IDNO.5和SEQ ID NO.6所示；检测TAC1的引物如SEQ ID NO.7和SEQ ID NO.8所示。
6.一种糖尿病肾病诊断或辅助诊断试剂盒，其特征在于包含定量检测MS4A6A、CPA3、CXCL6和TAC1中的任意两种或两种以上的试剂。
7.根据权利要求6所述的糖尿病肾病诊断或辅助诊断试剂盒，其特征在于所述的定量检测MS4A6A、CPA3、CXCL6和TAC1中的任意两种或两种以上的试剂为检测所述RNA的引物、探针或基因芯片。
8.根据权利要求7所述的糖尿病肾病诊断或辅助诊断试剂盒，其特征在于检测MS4A6A的引物如SEQ ID NO.1和SEQ ID NO.2所示；检测CPA3的引物如SEQ ID NO.3和SEQ ID NO.4所示；检测CXCL6的引物如SEQ ID NO.5和SEQ ID NO.6所示；检测TAC1的引物如SEQ IDNO.7和SEQ ID NO.8所示。
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