大動(dòng)脈粥樣硬化型缺血性卒中易感基因研究進(jìn)展

劉蘭 王為珍 王喬樹

腦卒中是嚴(yán)重威脅人類健康的疾病之一，居全球病死原因第2位，亦是成人主要病殘?jiān)颍?］。腦卒中是遺傳因素和環(huán)境因素共同作用的結(jié)果，其中遺傳因素在發(fā)病機(jī)制中起重要作用。近年來，缺血性卒中候選基因研究已成為腦卒中遺傳學(xué)機(jī)制的研究重點(diǎn)。根據(jù)TOAST分型，缺血性卒中可以分為5種類型，即大動(dòng)脈粥樣硬化型（LAA型）、心源性栓塞型（CE型）、小動(dòng)脈閉塞型（SAO型）、其他明確病因型（SOD型）和不明病因型（SUD型），其中，LAA型缺血性卒中系腦血管造影證實(shí)與缺血性卒中神經(jīng)功能缺損相對應(yīng)的顱內(nèi)或顱外大動(dòng)脈狹窄率＞50%或閉塞，且符合動(dòng)脈粥樣硬化改變。動(dòng)脈粥樣硬化是以脂質(zhì)代謝障礙、血管內(nèi)皮細(xì)胞功能障礙、炎性細(xì)胞浸潤致炎癥反應(yīng)、動(dòng)脈粥樣硬化斑塊破裂、最終形成血栓為特點(diǎn)的復(fù)雜慢性病理生理學(xué)過程［2］。基于上述發(fā)病機(jī)制探尋LAA型缺血性卒中基因治療靶點(diǎn)，近年國內(nèi)外學(xué)者已經(jīng)進(jìn)行大量研究揭示LAA型缺血性卒中的遺傳易感性，并結(jié)合其他危險(xiǎn)因素（如高血壓、糖尿病、高脂血癥和心臟病等）進(jìn)行評價(jià)，對指導(dǎo)臨床醫(yī)師建立更佳、更新的診斷與治療方法具有積極意義。本文擬對目前研究較多的LAA型缺血性卒中易感基因研究進(jìn)展進(jìn)行簡要綜述。

一、ApoE基因

載脂蛋白E（ApoE）是包含299個(gè)氨基酸的磷脂糖蛋白，主要存在于血液乳糜顆粒（CM）、極低密度脂蛋白（VLDL）、中密度脂蛋白（IDL）和部分高密度脂蛋白（HDL）中，對脂質(zhì)代謝和心血管相關(guān)疾病有決定性作用。ApoE基因定位于染色體19q13.2，由3597個(gè)核苷酸組成，含4個(gè)外顯子和3個(gè)內(nèi)含子。ApoE是多態(tài)性蛋白質(zhì)，包含3個(gè)常見異構(gòu)體即E2、E3和E4，分別編碼3種亞型即ApoE2、ApoE3和ApoE4。ApoE各亞型與不同脂蛋白受體相互作用，通過乳糜顆粒和極低密度脂蛋白與肝臟不同酶類結(jié)合，調(diào)節(jié)吸收和分解代謝過程以改變血清膽固醇水平。ApoE基因型與高密度脂蛋白和膽固醇水平密切相關(guān)，可以影響動(dòng)脈進(jìn)展和動(dòng)脈疾病進(jìn)程。研究顯示，E2等位基因是低回聲和潰瘍型頸動(dòng)脈斑塊的獨(dú)立危險(xiǎn)因素［3］；與其他基因型相比，E3/E3基因型是腦卒中保護(hù)因素，特別是女性患者，E3等位基因可能具有潛在的神經(jīng)保護(hù)作用［4］。根據(jù)LAA型缺血性卒中與ApoE基因之間的相關(guān)性評價(jià)其對頸動(dòng)脈內(nèi)?中膜厚度（IMT）的作用，共納入22項(xiàng)臨床試驗(yàn)計(jì)30 879例存在血管病或血管危險(xiǎn)因素的患者進(jìn)行Meta分析，其結(jié)果顯示，ApoE2基因型患者頸動(dòng)脈內(nèi)?中膜厚度最低，ApoE3基因型患者適中，ApoE4基因型患者最高，提示頸動(dòng)脈內(nèi)?中膜厚度增加與ApoE基因特異性表達(dá)有關(guān)，尤其與ApoE4基因型有關(guān)，并與環(huán)境因素（如高血壓、吸煙等）相互作用，增加腦卒中發(fā)病風(fēng)險(xiǎn)［5］。馬飛煜等［6］在 LAA 型缺血性卒中患者中發(fā)現(xiàn)，含E4等位基因的患者血清低密度脂蛋白（LDL）水平顯著高于含E3等位基因的患者；LAA型缺血性卒中患者E4等位基因和E3/E4基因型頻率高于對照組，E3等位基因和E3/E3基因型頻率低于對照組；E4等位基因可以升高血清低密度脂蛋白水平，與LAA型缺血性卒中相關(guān)，而與其他類型缺血性卒中無明顯關(guān)聯(lián)性。

二、MMP?12基因

基質(zhì)金屬蛋白酶（MMPs）是一組依賴金屬離子作為輔助因子催化降解細(xì)胞外基質(zhì)（ECM）的酶群。基質(zhì)金屬蛋白酶?12（MMP?12）是基質(zhì)金屬蛋白酶家族成員，可降解細(xì)胞外基質(zhì)蛋白，并在動(dòng)脈粥樣硬化中有關(guān)鍵作用。研究顯示，MMP?12 mRNA高表達(dá)可以促進(jìn)巨噬細(xì)胞侵襲［7］和血管新生［8］，并在斑塊中活性增強(qiáng)［9］。MMP?12基因多態(tài)性對腦卒中的作用可能通過脂質(zhì)代謝、遺傳因素、環(huán)境因素或炎癥反應(yīng)機(jī)制介導(dǎo)。MMP?12基因除降解細(xì)胞外基質(zhì)外，還可通過激活腫瘤壞死因子?α（TNF?α）或調(diào)節(jié)促炎性因子如單核細(xì)胞趨化蛋白?1（MCP?1），促使巨噬細(xì)胞募集至血管壁［10］。Traylor等［11］進(jìn)行全基因組相關(guān)性研究（GWAS）發(fā)現(xiàn)，MMP?12基因在頸動(dòng)脈斑塊中呈明顯過表達(dá)，MMP?12基因rs660599多態(tài)性與LAA型缺血性卒中有關(guān)，這種易感基因增加早發(fā)性缺血性卒中發(fā)病風(fēng)險(xiǎn)［12］。基于轉(zhuǎn)基因兔模型的動(dòng)物實(shí)驗(yàn)證實(shí)，MMP?12基因可以促進(jìn)動(dòng)脈粥樣硬化的發(fā)生，刺激脂肪條紋形成至纖維斑塊的進(jìn)展［13］。Johnson 等［14］予 ApoE 基因敲除小鼠模型選擇性MMP?12抑制劑RXP470.1，結(jié)果顯示，RXP470.1可以延緩動(dòng)脈粥樣硬化進(jìn)展，究其原因，RXP470.1通過增加平滑肌細(xì)胞/巨噬細(xì)胞比例，減少巨噬細(xì)胞凋亡，增加纖維帽厚度，減少壞死核心，降低鈣化，從而增加斑塊穩(wěn)定性。MMP?12基因表達(dá)變化與頸動(dòng)脈斑塊進(jìn)展、破裂［15］和晚期發(fā)育有關(guān)［16］，其轉(zhuǎn)錄水平影響頸動(dòng)脈斑塊穩(wěn)定性。基于中國漢族人群的研究顯示，MMP?12基因可能并非頸動(dòng)脈斑塊的危險(xiǎn)因素［17］。

三、HDAC9基因

組蛋白去乙酰化酶9（HDAC9）是對染色體結(jié)構(gòu)修飾和基因表達(dá)調(diào)控發(fā)揮重要作用的蛋白質(zhì)。HDAC9基因定位于染色體7p21.1，表達(dá)于動(dòng)脈內(nèi)膜和平滑肌細(xì)胞、顱內(nèi)血管、頸動(dòng)脈和冠狀動(dòng)脈等。研究顯示，HDAC9基因可以導(dǎo)致血管內(nèi)皮細(xì)胞損傷，是將腦損傷與表觀遺傳修飾相關(guān)聯(lián)的信號轉(zhuǎn)導(dǎo)通路的關(guān)鍵組成部分之一［18］。HDAC9蛋白可以抑制肌細(xì)胞生成，參與心臟發(fā)育，通過改變腦組織缺血性反應(yīng)增加腦卒中發(fā)病風(fēng)險(xiǎn)，并對神經(jīng)元存活有影響。HDAC9基因敲除可以導(dǎo)致脂質(zhì)平衡基因增加、炎癥基因減少，巨噬細(xì)胞在ATP結(jié)合盒轉(zhuǎn)運(yùn)子A1（ABCA1）、ATP結(jié)合盒轉(zhuǎn)運(yùn)子G1（ABCG1）和過氧化物酶增殖物激活受體γ（PPARγ）基因啟動(dòng)子處通過H3和H3K9乙酰化，組蛋白聚集，促進(jìn)巨噬細(xì)胞向M2型轉(zhuǎn)化。HDAC9基因表達(dá)上調(diào)可抑制膽固醇外排和活化巨噬細(xì)胞產(chǎn)生，促進(jìn)動(dòng)脈粥樣硬化［19］。2012年，英國和德國等歐洲國家進(jìn)行的一項(xiàng)全基因組相關(guān)性研究確定HDAC9基因與LAA型缺血性卒中的關(guān)系，此后多項(xiàng)國際多中心全基因組相關(guān)性研究均證實(shí)HDAC9基因多態(tài)性與LAA型缺血性卒中相 關(guān)［20?22］。 Markus 等［23］發(fā) 現(xiàn) ，HDAC9 基 因rs11984041和rs2107595多態(tài)性與無癥狀性頸動(dòng)脈斑塊和頸動(dòng)脈內(nèi)?中膜厚度增加相關(guān)，HDAC9 mRNA在頸動(dòng)脈斑塊中表達(dá)上調(diào)，這與加速動(dòng)脈粥樣硬化進(jìn)展機(jī)制相一致，而少見于其他動(dòng)脈，可能是通過加快斑塊進(jìn)展、促進(jìn)斑塊不穩(wěn)定，增加缺血性卒中發(fā)病風(fēng)險(xiǎn)。研究業(yè)已證實(shí)HDAC9基因是心肌梗死、冠心病的主要風(fēng)險(xiǎn)基因［24?25］。故推測 HDAC9 基因靶向抑制劑有可能預(yù)防動(dòng)脈粥樣硬化，成為LAA型缺血性卒中的有效 治療藥 物［19，24］。HDAC9 基因rs2107595多態(tài)性與歐洲人群LAA型缺血性卒中相關(guān)，但中國南方漢族人群腦卒中與rs2107595多態(tài)性無關(guān)聯(lián)性，而與血清總膽固醇和甘油三酯（TG）相關(guān)，可能是由于rs2107595位點(diǎn)的等位基因頻率不同造成歐洲和中國人群腦卒中風(fēng)險(xiǎn)差異［26］。第二軍醫(yī)大學(xué)附屬長海醫(yī)院共納入279例腦卒中患者和984例正常對照者，基因檢測顯示，LAA型缺血性卒中與HDAC9基因rs11984041多態(tài)性無關(guān)聯(lián)性，而與rs2389995 和rs2240419 多態(tài)性相關(guān)［27］。

四、NINJ2基因

神經(jīng)損傷誘導(dǎo)蛋白2（NINJ2）系由142個(gè)氨基酸組成、NINJ2基因編碼的黏附分子，在神經(jīng)系統(tǒng)發(fā)育、分化、再生過程中調(diào)節(jié)細(xì)胞之間或細(xì)胞與基質(zhì)之間的相互作用。NINJ2基因是腦損傷修復(fù)相關(guān)基因，是腦卒中相關(guān)單核苷酸多態(tài)性（SNP）基因，其產(chǎn)物是神經(jīng)損傷誘導(dǎo)蛋白，于神經(jīng)損傷后呈高表達(dá)，參與神經(jīng)修復(fù)和再生，表達(dá)于成熟感覺神經(jīng)元，促進(jìn)神經(jīng)軸突增生，使受損神經(jīng)遠(yuǎn)端施萬細(xì)胞數(shù)目增加［28］。NINJ2基因表達(dá)變化影響腦組織對缺氧缺血的耐受［29］。NINJ2基因啟動(dòng)子（rs3809263 G＞A）單核苷酸多態(tài)性是功能性的，是LAA型缺血性卒中的生物學(xué)標(biāo)志物［30］。Ikram等［29］的全基因組相關(guān)性研究顯示，臨近NINJ2基因染色體12p13區(qū)的rs11833579和rs12425791多態(tài)性與白種人腦卒中相關(guān)，特別是增加LAA型缺血性卒中的發(fā)病風(fēng)險(xiǎn)。王鋒等［31］對128例LAA型缺血性卒中患者和112例正常對照者進(jìn)行基因檢測，結(jié)果顯示，NINJ2基因rs11833579位點(diǎn)隱性模型（AA/AG基因型）與LAA型缺血性卒中密切相關(guān)，且在后循環(huán)動(dòng)脈粥樣硬化中的頻率顯著高于其他基因型，提示NINJ2基因多態(tài)性可能與動(dòng)脈粥樣硬化的責(zé)任血管有關(guān)，而且不同血管發(fā)生動(dòng)脈粥樣硬化的概率可能與遺傳因素密切相關(guān)。基于中國人群的研究顯示，NINJ2基因染色體12p13區(qū)rs11833579和rs12425791多態(tài)性與LAA 型缺血性卒中密切相關(guān)［31?33］，且 rs12425791 位點(diǎn) A 等位基因增加腦卒中發(fā)病風(fēng)險(xiǎn)［34?35］；亦有少數(shù)研究結(jié)果顯示，rs11833579多態(tài)性與腦卒中無關(guān)聯(lián)性［28，36］；在非洲裔美國人群和巴基斯坦人群中，NINJ2基因染色體12p13區(qū)rs11833579和rs12425791多態(tài)性與缺血性卒中無關(guān)聯(lián)性［37］。

五、CXCL16基因

CXC型趨化因子配體16（CXCL16）基因定位于染色體17p13區(qū)，包含5個(gè)外顯子。CXCL16蛋白是集趨化因子、黏附分子、清道夫受體為一體的免疫因子，與其受體共同表達(dá)于血管平滑肌細(xì)胞，在誘導(dǎo)大動(dòng)脈平滑肌細(xì)胞增殖、細(xì)胞間粘附、脂質(zhì)累積和基質(zhì)降解中發(fā)揮重要作用［38］。研究顯示，CXCL16基因通過促動(dòng)脈粥樣硬化因子、干擾素?γ（IFN?γ）以及炎癥反應(yīng)等促進(jìn)斑塊形成［39］和斑塊不穩(wěn)定［40］。CXCL16 mRNA表達(dá)于培養(yǎng)的動(dòng)脈內(nèi)皮細(xì)胞［41］，不僅可以活化血小板，而且可以促進(jìn)CXCL16蛋白成為有效的新型血小板黏附配體，誘導(dǎo)血小板粘附至血管壁［42］。Wang等［43］對 244 例 LAA 型缺血性卒中患者、153例存在動(dòng)脈粥樣硬化危險(xiǎn)因素但未發(fā)生腦卒中的患者和167例正常對照者進(jìn)行聚合酶鏈反應(yīng)?限制性片段長度多態(tài)性（PCR?RFLP），結(jié)果顯示，LAA型缺血性卒中患者CXCL16蛋白水平明顯升高，而3組患者CXCL16 p.Ala181Val基因型分布以及等位基因頻率差異無統(tǒng)計(jì)學(xué)意義。劉丹等［44］的研究顯示，LAA型缺血性卒中患者CXCL16 p.Ala181Val的AA基因型分布和A等位基因頻率明顯高于正常對照者，多因素Logistic回歸分析顯示，AA基因型是缺血性卒中的獨(dú)立危險(xiǎn)因素，表明CXCL16 p.Ala181Val多態(tài)性與LAA型缺血性卒中遺傳易感性相關(guān)，A等位基因是LAA型缺血性卒中的遺傳易感基因之一。亦有研究顯示，血清CXCL16蛋白水平與LAA型缺血性卒中和頸動(dòng)脈粥樣硬化相關(guān)［43，45］，測定 CXCL16 蛋白水平不僅有助于識別LAA型缺血性卒中高危患者，且與急性缺血性卒中不良預(yù)后相關(guān)［38，40］。CXCL16 基因參與和促進(jìn)動(dòng)脈粥樣硬化形成和進(jìn)展，與動(dòng)脈粥樣硬化性疾病如冠心病、頸動(dòng)脈粥樣硬化、腦卒中等的發(fā)病密切相關(guān)。

六、其他

染色體9p21.3區(qū)與LAA型缺血性卒中相關(guān)的主要單核苷酸多態(tài)性長度約100×103bp，該片段與INK4位點(diǎn)反義非編碼RNA（ANRIL）的外顯子18～24部分重疊［46］。ANRIL基因表達(dá)于人類動(dòng)脈粥樣硬化血管和頸動(dòng)脈內(nèi)膜，亦表達(dá)于血管內(nèi)皮細(xì)胞、單核細(xì)胞起源的巨噬細(xì)胞以及冠狀動(dòng)脈平滑肌細(xì)胞［46］，在動(dòng)脈粥樣硬化中發(fā)揮一定作用。染色體9p21.3區(qū)的遺傳片段不僅與甲硫腺苷磷酸（MTAP）基因剪接變體外顯子5進(jìn)一步重疊，而且臨近細(xì)胞周期蛋白依賴性激酶抑制基因2A/B（CDKN2A/B，分別表達(dá)p16INK4A和p15INK4B基因），上述基因在細(xì)胞增殖、衰老和凋亡中發(fā)揮重要作用。相關(guān)研究顯示，ANRIL、p16INK4A、p15INK4B、染色體9p21.3區(qū)共同協(xié)調(diào)轉(zhuǎn)錄［49］。2012年，METASTROKE協(xié)作組對多個(gè)全基因組相關(guān)性研究進(jìn)行Meta分析，證實(shí)染色體9p21.3區(qū)與LAA型缺血性卒中相關(guān)（r=1.150，P＜0.001）［21］；近年的多項(xiàng)研究均證實(shí)二者具有相關(guān)性［49?50］。染色體 9p21 區(qū) rs2383206 和 rs4977574 多態(tài)性與中國漢族人群頸動(dòng)脈斑塊潛在相關(guān)［51］，rs10757278多態(tài)性與女性頸動(dòng)脈斑塊呈正相關(guān)（r=2.420，P=0.013）［52］，rs1333035 多態(tài)性可能與斑塊破裂和血栓形成相關(guān)［53］。Musunuru 等［54］發(fā)現(xiàn)，染色體9p21.3區(qū)與血小板聚集明顯相關(guān)（P＜0.001），推測染色體9p21.3區(qū)可能通過調(diào)節(jié)血小板活性而增加斑塊破裂和血栓形成風(fēng)險(xiǎn)，從而導(dǎo)致本身已存在動(dòng)脈粥樣硬化的人群發(fā)生LAA型缺血性卒中。未來尚待進(jìn)一步確定LAA型缺血性卒中與染色體9p21.3區(qū)之間的聯(lián)系是否通過上述基因或其他可能途徑進(jìn)行遠(yuǎn)距離調(diào)節(jié)［46］。盡管目前業(yè)已證實(shí)染色體9p21.3區(qū)是冠狀動(dòng)脈疾病和心肌梗死的主要風(fēng)險(xiǎn)基因，但該基因和腦卒中的關(guān)系不依賴冠狀動(dòng)脈疾病、心肌梗死或者其他血管危險(xiǎn)因素而獨(dú)立發(fā)揮作用［25］。

綜上所述，腦卒中是多基因、多因素互相作用疾病。目前與其發(fā)病相關(guān)的基因研究大部分針對單基因，且國內(nèi)外報(bào)道多不盡一致，究其原因，主要有以下幾方面：（1）種族和人群差異。（2）大部分臨床研究樣本量較小，統(tǒng)計(jì)學(xué)說服力參差不齊。（3）對多基因遺傳性疾病，單一基因作用較小，易受其他基因和環(huán)境的影響。（4）不同的入組標(biāo)準(zhǔn)存在選擇偏倚。因此，為準(zhǔn)確篩選LAA型缺血性卒中候選基因，尚待更大樣本量，同時(shí)開展遺傳流行病學(xué)和分子流行病學(xué)調(diào)查，以及綜合考慮多種因素。通過腦卒中易感基因研究，使臨床醫(yī)師可以從遺傳學(xué)角度篩查腦卒中高危人群，早期預(yù)防疾病發(fā)生；也可以從遺傳學(xué)角度對腦卒中進(jìn)行病因分型，針對不同患者的個(gè)體化治療將是未來腦卒中基因研究的重點(diǎn)。

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