CN110468196A - 一种预测氯吡格雷抵抗的生物标志物的应用及其试剂盒 - Google Patents

一种预测氯吡格雷抵抗的生物标志物的应用及其试剂盒 Download PDF

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CN110468196A
CN110468196A CN201910769174.7A CN201910769174A CN110468196A CN 110468196 A CN110468196 A CN 110468196A CN 201910769174 A CN201910769174 A CN 201910769174A CN 110468196 A CN110468196 A CN 110468196A
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clopidogrel
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谢红光
李逸飞
吉金子
邰婷
米琼宇
谢文剑
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Puling Biological Nanjing Co ltd
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Abstract

本发明公开了一种预测氯吡格雷抵抗的生物标志物的应用及其试剂盒。人外周血白细胞mARC1mRNA的相对表达量作为检测靶标在制备预测氯吡格雷抵抗的试剂盒中的应用。一种预测氯吡格雷抵抗的试剂盒，包含检测人外周血白细胞mARC1mRNA表达的引物对及其序列和其它相关试剂。在临床上，通过对人外周血白细胞mARC1mRNA相对表达量的检测可预测氯吡格雷抵抗，为指导临床合理用药、帮助临床医生诊断或预测氯吡格雷的临床疗效和预后提供了全新的检测方法。

Description

一种预测氯吡格雷抵抗的生物标志物的应用及其试剂盒
技术领域
本发明属于生物检测领域，涉及一种预测氯吡格雷抵抗的生物标志物的应用及其试剂盒，具体涉及人外周血白细胞mARC1 mRNA作为预测氯吡格雷抵抗的生物标志物及其应用。
背景技术
作为一种已经入选WHO基本药物目录的抗血小板药物，氯吡格雷被用来预防急性冠脉综合征(ACS)心肌缺血的再次发生或预防经皮冠脉介入术(PCI)后冠脉支架内血栓的形成，与阿司匹林合用所组成的“双抗血小板治疗”方案是ACS或PCI植入支架后临床抗血小板治疗方案的“金标准”，为世界各国广泛采用。
氯吡格雷抵抗(或耐受)是指病人在服用推荐(或标准)剂量的氯吡格雷后因各种原因不能达到预期的抗血小板作用而导致的药物临床疗效较差或无效的现象。据估计，约10％–45％的服药病人可能存在氯吡格雷抵抗。判断服药病人有无氯吡格雷抵抗的常用方法之一是用服药前后ADP诱导的血小板聚集抑制率的改变或用药后的血小板高反应性来反映氯吡格雷抵抗的有无或程度。然而，目前各种检测血小板聚集率方法的重现性较差，难以精准指导临床合理使用氯吡格雷。
此外，目前临床采用的病人外周血白细胞DNA检测CYP2C19基因型也被视为指导病人合理用药的依据之一。但是，CYP2C19基因型的个体间差异仅能反映病人在服用氯吡格雷后临床疗效个体差异的12％。我们最近的临床研究发现，用CYP2C19基因型区分氯吡格雷抵抗与否的诊断敏感性仅为30.4％，ROC曲线下面积是0.617，显示其临床诊断价值或预测意义十分有限。病人产生氯吡格雷抵抗的原因较多，但其相关机制不甚明了，临床上仍缺乏用之有效的预测氯吡格雷抵抗的生物标志物。
钼依赖性线粒体胺肟基还原组分1(mitochondrial amidoxime-reducingcomponent 1,mARC1)是近年发现的一种能高效还原所有含氮化合物(或N位被氧化代谢产物)的细胞线粒体酶。新近研究发现，mARC1能还原三甲胺氮氧化物(TMAO)成它的前体三甲胺(TMA)，也能还原硝酸盐成一氧化氮(NO)。已知TMAO能诱导血小板聚集，而NO能抑制血小板聚集。我们最近发现，氯吡格雷能上调mARC1表达(未发表数据)。据此推测，口服氯吡格雷后，不同个体mARC1表达水平的差异可预测氯吡格雷抵抗。
发明内容
本发明的目的是针对现有相关技术的上述不足，提供一种全新的预测氯吡格雷抵抗的生物标志物及其应用。
本发明的目的可通过以下技术方案来实现：
人外周血白细胞mARC1 mRNA水平作为检测靶标在制备预测氯吡格雷抵抗的试剂盒中的应用。
检测人外周血白细胞中mARC1 mRNA的试剂在制备预测氯吡格雷抵抗的试剂盒中的应用。
一种预测氯吡格雷抵抗的试剂盒，包含检测人外周血白细胞中mARC1 mRNA相对表达量的引物对及其序列。
所述的检测人外周血白细胞中mARC1 mRNA相对表达量的试剂，优选包括qRT-PCR法检测人外周血白细胞中mARC1 mRNA相对表达量的相关试剂。
有益效果
本发明发现了人外周血白细胞中mARC1 mRNA相对表达量的高低能准确预测氯吡格雷抵抗(见图1)。基于上述发现，本发明提供了一种可预测氯吡格雷抵抗的全新的生物标志物，即人外周血白细胞中mARC1 mRNA的相对表达量，该标志物作为检测靶标可预测或辅助判断病人服用氯吡格雷后的临床疗效或预后。临床研究结果显示：人外周血白细胞中mARC1 mRNA相对表达量检测对诊断氯吡格雷抵抗的ROC曲线下面积是0.698，敏感性是62.5％，特异性为71.3％(见图2)。
附图说明
图1.人外周血白细胞中mARC1 mRNA相对表达量及其在氯吡格雷敏感者(S)和抵抗者(R)的组间差异(mean±SEM；1.29±0.11vs.0.72±0.05；S vs.R；P<0.0001)。
图2.人外周血白细胞中mARC1 mRNA诊断氯吡格雷抵抗的ROC曲线。
具体实施方式
人外周血白细胞中mARC1 mRNA相对表达量检测在诊断氯吡格雷抵抗中的应用
一、检测人白细胞中mARC1 mRNA相对表达量的试剂盒，包括以下成分：
红细胞裂解液
TRIzol LS试剂；
三氯甲烷；
异丙醇；
DEPC水
gDNA eraser
5×gDNA eraser buffer
RT enzyme mix I
buffer 2(for Real-Time)
RT primer mix
RNase-free dH2O
Premix Ex TaqTM II(2×)
ROX Reference Dye II(50×)
针对mARC1mRNA检测的特异性引物对序列SEQ ID NO.1和SEQ ID NO.2。
mRNA定量分析时所需内参照GAPDH的引物对(SEQ ID NO.3和SEQ ID NO.4)。
二、试剂盒使用方法
测定人外周血白细胞mARC1 mRNA相对表达量具体操作如下：
1、人外周血白细胞总RNA的提取：
(1)取1mL枸橼酸钠抗凝的人全血，加入等量红细胞裂解液，涡旋混匀，室温静置15min，600×g离心，弃上清得人白细胞；
(2)向制得的人白细胞中加入1mL TRIzol，涡旋混匀，室温静置5min；
(3)加入0.2mL三氯甲烷，涡旋混匀，室温静置10min；
(4)12000×g，4℃离心15min；
(5)吸取含有总RNA的上清，转移至新的1.5mL EP管中，加入等体积异丙醇后颠倒混匀，室温下静置10min；
(6)12000×g，4℃离心10min，弃上清；
(7)加入1mL 75％乙醇(由DEPC水配制)涡旋震荡混匀；
(8)7500×g，4℃离心5min，弃上清；
(9)室温放置，挥干RNA沉淀，约10min；
(10)待RNA无明显液滴时，加10μL RNase-free dH2O(或DEPC水)，冰上静置10min
(11)混匀，置冰上备用。
2、人白细胞mRNA逆转录：
(1)基因组DNA的去除反应
反应条件为42℃ 2min，4℃保存。
(2)逆转录反应(生成其cDNA)
反应条件为37℃ 15min，85℃ 5s，4℃保存。
3、qRT-PCR法测定人白细胞中mARC1mRNA的相对表达量:
实时荧光定量PCR(应用ABI7500Real-Time PCR System的操作方法)检测mARC1的cDNA水平，采用SYBR Premix Ex TaqTM(TaKaRa Biotech Co.,Ltd.,Dalian,China)荧光定量PCR试剂。
引物设计见表1，反应体系20μL，包含：
PCR反应条件：预变性：95℃ 30s；PCR反应：95℃ 5s，60℃ 34s，共40个循环。溶解曲线：95℃ 15s，60℃ 1min，95℃ 15s。
荧光定量PCR的结果以每个反应管内的荧光信号达到设定的阈值时所经历的循环数(Ct值表示)。ΔCt＝Ct(mARC1)–Ct(GAPDH)，结果采用2-ΔΔCt计算mARC1的mRNA相对表达量。
表1.qRT-PCR引物及其序列
三、有效性
选取182名经PCI植入药物涂层支架后进行冠状动脉造影复查的病人，口服氯吡格雷75mg/天长达至少6个月。造影前抽取病人早晨空腹静脉血1管(约1mL)，枸橼酸抗凝(蓝盖管)用于ADP诱导的全血血小板聚集率测定，剩余全血提取白细胞，冻存于–80℃冰箱，备用。
根据测得的全血血小板聚集率将病人分为两组，其中：
阴性组：对氯吡格雷反应敏感者(S)，血小板聚集率为0–4欧姆，共88名；
阳性组：对氯吡格雷反应不敏感者(R)，血小板聚集率为≥5欧姆，共92名。
根据上述实验方法提取病人外周血白细胞mRNA，用上述方法检测其中mARC1mRNA的相对表达量。本发明以88名氯吡格雷反应敏感者白细胞中mARC1mRNA相对表达量作为(氯吡格雷抵抗)阴性组，以92名氯吡格雷反应不敏感者白细胞中mARC1mRNA相对表达量作为作为(氯吡格雷抵抗)阳性组，分别检测并比较两组之间白细胞中mARC1mRNA相对表达量的组间差异(见图1)；建立人外周血白细胞中mARC1mRNA相对表达量诊断氯吡格雷抵抗的ROC曲线(见图2)。结果显示，ROC曲线下面积(AUC)为0.698(95％CI:0.622–0.774；P<0.0001)。对氯吡格雷抵抗诊断的敏感性(真阳性率)为62.5％，特异性(真阴性率)为71.3％，约登(Youden)指数J为0.338，诊断氯吡格雷抵抗的最佳临界(cutoff)值为0.885。

Claims (4)

1.人外周血白细胞mARC1 mRNA的相对表达量作为检测靶标在制备预测氯吡格雷抵抗的试剂盒中的应用。
2.一种预测氯吡格雷抵抗的全新试剂盒，其特征在于包含检测人外周血白细胞mARC1mRNA相对表达量的引物对及其序列。
3.检测人外周血白细胞mARC1 mRNA相对表达量的相关试剂在制备预测氯吡格雷抵抗的试剂盒中的应用。
4.根据权利要求2所述的试剂盒，其特征在于所述的检测人白细胞mARC1mRNA相对表达量的引物及其相关试剂。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113274504A (zh) * 2021-06-30 2021-08-20 南京市第一医院 作用于胆碱或tmao相关靶点的化合物在制备预防和/或逆转氯吡格雷抵抗药物中的应用

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CN107541562A (zh) * 2016-06-29 2018-01-05 南京市第医院 ABCC3-013 mRNA在制备检测氯吡格雷抵抗的试剂盒中的应用及其试剂盒

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CN107541562A (zh) * 2016-06-29 2018-01-05 南京市第医院 ABCC3-013 mRNA在制备检测氯吡格雷抵抗的试剂盒中的应用及其试剂盒

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113274504A (zh) * 2021-06-30 2021-08-20 南京市第一医院 作用于胆碱或tmao相关靶点的化合物在制备预防和/或逆转氯吡格雷抵抗药物中的应用

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