CYP2C19基因多態(tài)性及患者主要臨床資料與氯吡格雷藥物抵抗的相關(guān)性研究

姜金坪，杜曉明，孫亞欣，馬淑梅*

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CYP2C19基因多態(tài)性及患者主要臨床資料與氯吡格雷藥物抵抗的相關(guān)性研究

姜金坪a，杜曉明b，孫亞欣b，馬淑梅a*

目的觀察藥物代謝酶系統(tǒng)中CYP2C19基因多態(tài)性及患者主要臨床資料與服用氯吡格雷前后血小板聚集率變化(氯吡格雷藥物抵抗)的相關(guān)性。方法入選擬行冠脈造影檢查或支架植入治療患者35例，根據(jù)圍手術(shù)期應(yīng)用氯吡格雷前后血小板聚集率變化，將患者分為氯吡格雷抵抗組和非抵抗組。檢測(cè)CYP2C19基因型，并記錄患者年齡、性別、煙酒史、高血壓、糖尿病等主要臨床資料，分析基因水平及臨床水平各因素對(duì)血小板聚集及氯吡格雷藥物抵抗的影響。結(jié)果檢測(cè)出氯吡格雷抵抗的患者15例，CYP2C19慢代謝基因型患者4例，Logistic回歸分析顯示，CYP2C19基因型是氯吡格雷抵抗的危險(xiǎn)因素(OR=1.236，95%CI：0.273～5.599，P=0.049)。結(jié)論CYP2C19基因型在基因水平與氯吡格雷抵抗相關(guān)，臨床水平資料未見(jiàn)明顯相關(guān)性。

血小板聚集功能；氯吡格雷抵抗；CYP2C19基因多態(tài)性

0　引言

阿司匹林及氯吡格雷雙聯(lián)抗血小板治療是冠心病心絞痛及急性冠脈綜合征住院患者圍手術(shù)期的常規(guī)治療方法[1]。隨著氯吡格雷抵抗及新型ADP P2Y12受體抑制劑(普拉格雷、替格瑞羅)的出現(xiàn)，針對(duì)不同患者實(shí)施個(gè)體化治療越來(lái)越被關(guān)注[2-3]。因?yàn)镃YP2C19基因多態(tài)性與支架內(nèi)血栓及再發(fā)心肌梗死密切相關(guān)，CYP2C19基因多態(tài)性被認(rèn)為是導(dǎo)致氯吡格雷抵抗在基因水平的影響因素[4-12]。臨床可應(yīng)用ADP誘導(dǎo)血小板聚集來(lái)衡量氯吡格雷應(yīng)用后對(duì)血小板聚集功能抑制的有效性，本文于用藥前及用藥后分別檢測(cè)血小板聚集功能，根據(jù)藥物應(yīng)用前后血小板聚集率變化來(lái)定義氯吡格雷抵抗組及非抵抗組。現(xiàn)報(bào)道如下。

1　對(duì)象與方法

1.1研究對(duì)象選取2014年12月至2015年1月入住我院預(yù)計(jì)接受冠脈造影檢查或支架植入治療患者35例。所有入選患者入院前均未服用過(guò)阿司匹林、氯吡格雷等抗血小板治療藥物，患者年齡18～85歲，性別不限，主要臨床診斷為冠心病、穩(wěn)定型心絞痛、急性冠脈綜合征均可。入院即檢測(cè)用藥前血小板聚集功能，急診手術(shù)患者給予阿司匹林及氯吡格雷負(fù)荷量各300 mg，至少6 h后檢測(cè)用藥后血小板聚集功能；擇期手術(shù)患者接受阿司匹林100 mg/d、氯吡格雷75 mg/d口服，至少7 h后檢測(cè)用藥后血小板聚集功能。

1.2研究方法

1.2.1血小板聚集功能檢測(cè)應(yīng)用南京神州英諾華醫(yī)療科技有限公司研發(fā)的PL-11自動(dòng)化血小板聚集功能檢測(cè)儀測(cè)定血小板聚集率。靜脈采血，枸櫞酸鈉1∶9比例抗凝;取全血標(biāo)本0.5 mL加0.9%生理鹽水0.5 mL，放置在試驗(yàn)槽內(nèi)，10 min后進(jìn)行測(cè)定[13]。Muller[14]定義最大血小板聚集基線水平與服用氯吡格雷后最大血小板聚集率變化<10%為無(wú)反應(yīng)，10%～29%為半反應(yīng)，>30%為正常反應(yīng)。本試驗(yàn)將用藥前后血小板聚集率變化率<30%定義為氯吡格雷藥物抵抗組，變化率≥30%定義為非抵抗組。

1.2.2CYP2C19基因型檢測(cè)應(yīng)用上海百傲科技有限公司生產(chǎn)的CYP2C19基因檢測(cè)芯片試劑盒進(jìn)行CYP2C19基因型多態(tài)性檢測(cè)。靜脈采血2 mL，加至EDTA抗凝管中，并充分混勻，血液標(biāo)本采集后及時(shí)檢測(cè)，操作流程包括樣本DNA抽提-PCR擴(kuò)增-雜交檢測(cè)等。通過(guò)分離外周血淋巴細(xì)胞基因組DNA，分別用一對(duì)等位基因特異性的反向引物與共用正向引物進(jìn)行PCR擴(kuò)增，通過(guò)電泳分析對(duì)應(yīng)的PCR產(chǎn)物來(lái)判斷CYP2C19基因型。CYP2C19等位基因主要是*1、*2、*3型，CYP2C19*2和CYP2C19*3是基因突變的主要類(lèi)型。野生型(*l/*1)為快代謝型，突變雜合型(*1/*2，*1/*3)為中間代謝型，突變純合型(*2/*2，*2/*3，*3/*3)為慢代謝型。

1.2.3患者主要臨床資料記錄所有研究對(duì)象性別、年齡、體重指數(shù)(BMI)、吸煙及飲酒史、高血壓及糖尿病病史、膽固醇、三酰甘油、高密度脂蛋白、低密度脂蛋白、糖化血紅蛋白、血肌酐、血尿酸、血小板計(jì)數(shù)、纖維蛋白原、D-二聚體等臨床資料。

2　結(jié)果

2.1CYP2C19基因型分布入選35例患者中，CYP2C19基因型分布為快代謝基因型15例(42.86%)，中間代謝基因型16例(45.71%)，慢代謝基因型4例(11.43%)，見(jiàn)表1。

2.2血小板聚集功能變化率患者用藥前血小板最大聚集率為55.94%±15.62%，用藥后血小板最大聚集率為34.83%±17.07%；用藥前后聚集功能變化率為37.73%±25.15%。其中聚集功能變化率<30%(氯吡格雷藥物抵抗)15例(42.86%)，變化率≥30%(非抵抗)20例(57.14%)。

2.3主要臨床指標(biāo)統(tǒng)計(jì)入選35例患者中，男20例，女15例，年齡26～80歲，平均59歲，兩組患者的臨床資料見(jiàn)表2。對(duì)存在差異項(xiàng)目進(jìn)行Logistic回歸分析，只有CYP2C19基因型(OR=1.236；95%CI：0.273～5.599；P=0.049<0.05)是氯吡格雷抵抗的危險(xiǎn)因素，具體見(jiàn)表3。且與*1/*1基因型相比，*1/*2,*1/*3基因型OR=1.846，*2/*2基因型OR=4.846。

表1　CYP2C19基因型結(jié)果統(tǒng)計(jì)(例，%)

表2　入選患者一般資料(例，%)

表3　Logistic回歸分析

3　討論

本研究觀察了CYP2C19基因多態(tài)性及主要臨床資料對(duì)服用氯吡格雷前后血小板聚集功能變化率(氯吡格雷抵抗)的影響。并通過(guò)Logistic回歸篩選出CYP2C19基因多態(tài)性是氯吡格雷抵抗的高危因素。

慢代謝基因型氯吡格雷抵抗發(fā)生率高，而快代謝基因型中也存在氯吡格雷抵抗，可見(jiàn)CYP2C19基因多態(tài)性只是氯吡格雷抵抗基因水平的一個(gè)影響因素(其他基因還有ABCB1、PON1)，而絕不是唯一影響因素[15-22]。有研究表明，在臨床水平糖尿病或糖化血紅蛋白影響血小板聚集功能[23-24]，而在本研究中未得到證實(shí)；在分子水平有藥物的相互作用(奧美拉唑)、血小板釋放的其他因子(5羥色胺、腎上腺素等)影響氯吡格雷藥物抵抗。因此，氯吡格雷抵抗存在基因水平、分子水平、臨床水平等多方面影響因素。

目前，臨床尚未確定血小板聚集功能檢測(cè)的金標(biāo)準(zhǔn)，因此，臨床上根據(jù)服藥后血小板聚集率，將患者分為氯吡格雷藥物抵抗組及非抵抗組并不合理，而本研究通過(guò)用藥前后血小板聚集率的變化率進(jìn)行分組，更加準(zhǔn)確地反映出個(gè)體對(duì)氯吡格雷藥物的反應(yīng)性。但本實(shí)驗(yàn)因需測(cè)量服藥前后兩次血小板聚集功能，操作起來(lái)相對(duì)困難，研究對(duì)象偏少，實(shí)驗(yàn)數(shù)據(jù)可能存在偏倚。隨著大家對(duì)氯吡格雷抵抗研究的深入，以及血小板聚集功能檢測(cè)方法的增加，相信氯吡格雷抵抗在分子水平方面的研究將有更廣闊的空間。

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Correlation of CYP2C19 gene polymorphism and major clinical data with clopidogrel drug resistance

JIANG Jin-pinga,DU Xiao-mingb,SUN Ya-xinb,MA Shu-meia*

(a.Department of Cardiology,b.Department of Pharmacy,Shengjing Hospital of China Medical University,Shenyang 110004,China)

ObjectiveTo observe the correlation of CYP2C19 gene polymorphism in drug metabolism enzyme system and main clinical data with the change of platelet aggregation rate before and after taking clopidogrel (clopidogrel drug resistance).MethodsTotally 35 patients with coronary angiography examination or stenting treatment were selected and divided into clopidogrel resistance group (resistance group) and the non-resistance group according to the change of platelet aggregation rate before and after taking clopidogrel.CYP2C19 genotype was tested,and the patients′age,sex,alcohol tobacco history,hypertension,diabetes and other major clinical data were recorded,and the effect of genetic level and clinical level on platelet aggregation and clopidogrel drug resistance were analyzed.ResultsThere were 15 cases with clopidogrel resistance and 4 cases with CYP2C19 genotype slow metabolism.Logistic regression analysis showed that CYP2C19 genotype was a risk factor for clopidogrel resistance (OR=1.236,95%CI:0.273～5.599,P=0.049).ConclusionCYP2C19 gene polymorphisms are associated with clopidogrel resistance at the genetic level,but clinical level shows no obvious correlation.

Platelet aggregation function;Clopidogrel resistance;CYP2C19 gene polymorphisms

2016-01-18

中國(guó)醫(yī)科大學(xué)附屬盛京醫(yī)院a.第一心血管內(nèi)科,b.藥學(xué)部,沈陽(yáng) 110004

10.14053/j.cnki.ppcr.201609010