湯永志 陳巧玲 江挺 朱敏 燕飛 陳華忠 朱堅(jiān)勝
Rs4129009基因多態(tài)性與慢性HBV感染的相關(guān)性研究
湯永志 陳巧玲 江挺 朱敏 燕飛 陳華忠 朱堅(jiān)勝
目的探討Toll樣受體10(TLR10)基因多態(tài)性位點(diǎn)Rs4129009(Ile775Val)與慢性HBV感染的關(guān)系。方法采集428例慢性HBV感染患者、210例健康對(duì)照人群外周血并提取全基因組DNA。采用聚合酶鏈反應(yīng)(PCR)法擴(kuò)增特定基因多態(tài)性位點(diǎn),產(chǎn)物經(jīng)純化后直接測序分析。結(jié)果兩組均符合Hardy-Weinberg平衡(均P>0.05),即研究對(duì)象具有群體代表性。與健康對(duì)照組比較,HBV感染組變異型(AG+GG)、等位基因G頻率均較低(均P<0.01)。高病毒載量(>106IU/ml)、HBeAg陽性的變異型基因比例較高(均P<0.01),而HBV感染程度、乙肝家族史與基因多態(tài)性無關(guān)(均P>0.05)。結(jié)論TLR10基因Rs4129009可能與慢性HBV感染相關(guān)。
Toll樣受體10基因多態(tài)性乙型肝炎病毒
慢性HBV感染嚴(yán)重威脅人類健康,每年約有100萬人死于HBV感染所致的肝衰竭、肝硬化和肝細(xì)胞癌等[1]。Toll樣受體2(TLR2)與慢性HBV感染關(guān)系密切,TLR2及其信號(hào)通路可作為抑制HBV復(fù)制的途徑,也可成為HBV發(fā)生免疫逃避的作用靶點(diǎn)[2]。TLR10是一類缺乏特異性配體的孤兒受體,已有文獻(xiàn)報(bào)道TLR10基因多態(tài)性Rs4129009(Ile775Val)與前列腺癌[3-4]、哮喘[5]、自身免疫性甲狀腺疾病[6]等的發(fā)生風(fēng)險(xiǎn)存在關(guān)聯(lián)。TLR10基因多態(tài)性Rs4129009位點(diǎn)在肝硬化失代償期合并感染后急性加重,或在慢加急性肝衰竭中促進(jìn)炎癥因子應(yīng)答,最終降低生存率[7]。TLR10與TLR2異源化形成二聚體或競爭獲取IL-1受體激動(dòng)劑,從而負(fù)性調(diào)節(jié)TLR2下游產(chǎn)生IL-1[8]。然而,TLR10基因多態(tài)性與慢性HBV感染是否相關(guān)未見報(bào)道;故筆者就TLR10外顯子區(qū)Rs4129009(Ile775Val)多態(tài)性與慢性HBV感染的關(guān)系作一探討,現(xiàn)報(bào)道如下。
1.1 對(duì)象選擇2012年9月至2015年12月在溫州醫(yī)科大學(xué)附屬臺(tái)州醫(yī)院因慢性乙型肝炎活動(dòng)首次住院的428例患者為HBV感染組,其中男280例,女148例;年齡18~73(41.8±11.8)歲;所有患者符合2010年《慢性乙型肝炎防治指南》的診斷標(biāo)準(zhǔn),并排除其他類型病毒肝炎、自身免疫性疾病、感染、藥物、飲酒等引起肝功能異常。選擇同期在該院體檢中心健康體檢的210例健康者為健康對(duì)照組,其中男134例,女76例;年齡22~69(42.0±10.1)歲;排除糖尿病、脂肪肝、自身免疫性疾病等體檢異常。兩組研究對(duì)象均為浙江地區(qū)漢族人,年齡、性別比較差異均無統(tǒng)計(jì)學(xué)意義(均P>0.05)。本研究通過醫(yī)院倫理委員會(huì)批準(zhǔn)。
1.2 試劑和設(shè)備血液基因組DNA提取試劑盒(1408G26,上海捷瑞生物工程有限公司);PCR引物(F:141124B85/R:141124B86,上海捷瑞生物工程有限公司)。核酸定量分析儀(1702525,美國Bio-rad公司);小型高速離心機(jī)(5418,德國Eppendorf公司);DNA測序儀(3730XL,美國ABI公司)。
1.3 方法
1.3.1 標(biāo)本采集清晨采集研究對(duì)象的空腹靜脈血2~3ml;血液標(biāo)本管含EDTA-Na抗凝,用于外周血基因組DNA提取;置于-80℃保存?zhèn)溆谩?/p>
1.3.2 TLR10外顯子區(qū)Rs4129009基因多態(tài)性檢測(1)DNA提?。菏褂醚夯蚪MDNA抽提試劑盒提取全血基因組DNA,采用核酸定量分析儀檢測DNA濃度及A260/A280比值,比值在1.7~2.0之間的樣本作為PCR擴(kuò)增模板。(2)PCR擴(kuò)增TLR10外顯子區(qū)上游引物:5′-CCAACTTTGTCCAGAATGAGTG-3′;下游引物5′-AGTGTATGTGGTCCCCAACTTC-3′。PCR擴(kuò)增體系:10×緩沖液2.5μl,DNA模板0.5μl,MgCl21.0μl(25mmol/L),dNTP 0.5μl,TaqDNA聚合酶0.25μl,引物各0.5μl,總體積為25μl,剩余體積用雙蒸水補(bǔ)足。PCR反應(yīng)條件:95℃預(yù)變性2min,94℃變性45s,56℃退火90s,72℃延伸2min;共循環(huán)35次;吸取擴(kuò)增產(chǎn)物5μl,加入溴化乙錠染色的1.5%瓊脂糖凝膠中電泳,凝膠成像分析PCR產(chǎn)物特異性。(3)DNA測序:PCR擴(kuò)增產(chǎn)物采用ABI3730XL測序儀進(jìn)行基因序列分析,由上海桑尼生物科技有限公司完成。
1.4 統(tǒng)計(jì)學(xué)處理應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件。兩組Rs4129009錯(cuò)義突變位點(diǎn)進(jìn)行Hardy-Weinberg平衡檢驗(yàn),以驗(yàn)證其群體代表性。兩組基因型及等位基因頻率[等位基因頻率=(純合子數(shù)×2+雜合子數(shù))/(受檢人群×2)]比較采用χ2檢驗(yàn)。兩組年齡比較采用獨(dú)立樣本t檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 擴(kuò)增測序結(jié)果HBV感染組與健康對(duì)照組PCR產(chǎn)物及基因測序PCR擴(kuò)增產(chǎn)物長度為383bp,擴(kuò)增片段測序結(jié)果見圖1。
圖1 TLR10基因Rs4129009位點(diǎn)的序列圖譜(a:野生純合子AA;b:變異純合子GG;c:變異雜合子AG)
2.2 兩組基因型及等位基因頻率比較經(jīng)檢驗(yàn),兩組均符合Hardy-Weinberg平衡(χ2=1.12、3.02,均P>0.05),表明研究對(duì)象具有群體代表性。HBV感染組AG+GG(變異型)頻率為44.2%(189/428),低于健康對(duì)照組的60.5%(127/210),差異有統(tǒng)計(jì)學(xué)意義(P<0.01);HBV感染組等位基因G頻率為25.9%(222/856),明顯低于健康對(duì)照組的35.0%(147/420),差異有統(tǒng)計(jì)學(xué)意義(P<0.01),見表2。
表2 Rs4129009基因型及等位基因的分布特征(個(gè))
2.3 HBV感染臨床特征與基因多態(tài)性的關(guān)系根據(jù)2000年中華醫(yī)學(xué)會(huì)傳染病與寄生蟲病學(xué)會(huì)和肝病學(xué)會(huì)修訂的《病毒性肝炎防治方案》將慢性HBV感染分為輕度、中度和重度,經(jīng)檢驗(yàn)發(fā)現(xiàn)不同HBV感染程度的基因多態(tài)性比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05);根據(jù)HBV DNA載量分成<103IU/ml、103~106IU/ml、>106IU/ml,經(jīng)檢驗(yàn)發(fā)現(xiàn)>106IU/ml組變異型比例較高(P<0.01),提示攜帶高病毒載量人群的變異型比例較高;此外,HBeAg陽性患者變異型比例較高(P<0.01),乙肝家族史與基因多態(tài)性無關(guān)(P>0.05),見表3。
表3 HBV感染臨床特征與基因多態(tài)性的關(guān)系(例)
TLR是哺乳動(dòng)物和昆蟲體內(nèi)一類進(jìn)化保守的I型跨膜蛋白,廣泛分布于多種細(xì)胞,能識(shí)別特定微生物表面的分子結(jié)構(gòu)——病原微生物相關(guān)分子模式(PAMPs),參與機(jī)體固有免疫和適應(yīng)性免疫應(yīng)答。人類TLR是一類高度保守的Toll樣蛋白,與果蠅的Toll樣蛋白同源,目前已確定有10種TLR在人類細(xì)胞上表達(dá)[9-10]。Wieland等[11]報(bào)道TLR3配體誘導(dǎo)產(chǎn)生IFN-β并抑制HBV復(fù)制;但在慢性HBV感染患者中TLR3表達(dá)及分泌功能受損[12],經(jīng)抗病毒治療后可恢復(fù);抗病毒應(yīng)答不佳或復(fù)發(fā)時(shí),TLR3表達(dá)再次下降。TLR2與TLR4擁有共同的MyD88信號(hào)通路,其下游分泌的IL-6、TNF-α能抑制原代肝細(xì)胞中HBV的復(fù)制[13-14];而相較于HBeAg陰性患者和健康人群,HBeAg陽性患者在肝細(xì)胞、庫弗細(xì)胞和外周單個(gè)核細(xì)胞中TLR2表達(dá)水平明顯下調(diào)[15]。HBV也可降低TLR2表達(dá)或抑制其信號(hào)通路以逃避免疫應(yīng)答,且TLR2表達(dá)與肝臟炎癥壞死成正比,在中重度肝炎、肝硬化和肝衰竭中可檢測到TLR2表達(dá)明顯增加,提示TLR2在肝炎和肝硬化的發(fā)生、發(fā)展中也起著重要作用[2]。TLR10位于4號(hào)染色體短臂1區(qū)3帶,包含4個(gè)外顯子,與TLR2、TLR1、TLR6形成基因簇,具體生物學(xué)功能尚未完全闡明,TLR10的配體至今未被確定,且缺乏經(jīng)典的下游信號(hào)通路[16],是TLR家族中的一類孤兒受體。TLR2與TLR1、TLR6形成同源或異源二聚體,參與針對(duì)大量細(xì)菌、真菌、病毒等病原微生物的免疫應(yīng)答,系統(tǒng)進(jìn)化樹分析表明TLR10與TLR2密切相關(guān);提示TLR10需要TLR2協(xié)助形成二聚體,從而募集MyD88,影響下游炎癥因子分泌以參與固有免疫應(yīng)答[16-17]。進(jìn)一步研究顯示TLR10和TLR2同時(shí)表達(dá)的細(xì)胞系信號(hào)轉(zhuǎn)導(dǎo)通路下游IL-8分泌下降,特異性抗人TLR10抗體阻斷TLR10后TLR2下游IL-1分泌上調(diào),提示TLR10為一負(fù)性調(diào)節(jié)受體[8],且TLR10對(duì)依賴MyD88、TRIF、MA-PK、IκB的信號(hào)通路及獨(dú)立的TLR通路也具有廣泛抑制效應(yīng)[18]。
TLR10基因單核苷酸多態(tài)性與人類疾病關(guān)系密切。一項(xiàng)多態(tài)性研究報(bào)道TLR10的2個(gè)SNPs位點(diǎn)(Rs11096955、Rs11096957)及TLR1基因上的多個(gè)SNPs位點(diǎn)均與前列腺癌發(fā)生密切相關(guān)[4]。亦有研究表明TLR10單核苷酸多態(tài)性位點(diǎn)Rs4129009(c.2322A>G)變異與哮喘發(fā)作風(fēng)險(xiǎn)相關(guān)[5]。另有報(bào)道EB病毒感染相關(guān)鼻咽癌、IgA腎病與TLR10的基因多態(tài)性位點(diǎn)也有一定關(guān)聯(lián)[19-20]。
Rs4129009是TLR10外顯子基因序列上的單核苷酸突變位點(diǎn)(G/A/T/C),不同堿基配對(duì)可導(dǎo)致蛋白表達(dá)改變;目前有研究表明Rs4129009(Ile775Val)基因多態(tài)性與前列腺癌[3-4]、哮喘[5]、自身免疫性甲狀腺疾病[6]等的發(fā)生風(fēng)險(xiǎn)存在關(guān)聯(lián)。為明確HBV感染與Rs4129009位點(diǎn)是否相關(guān),本研究比較并分析428例慢性HBV感染患者與210例健康人群的基因型,結(jié)果顯示HBV感染組AG+GG基因型頻率、G等位基因頻率與健康對(duì)照組比較,差異均有統(tǒng)計(jì)學(xué)意義;高病毒載量、HBeAg陽性的基因變異型比例較高,但與HBV感染程度、乙肝家族史均無關(guān)。
綜上所述,TLR2與慢性HBV感染關(guān)系密切,TLR2及其信號(hào)通路可作為抑制HBV復(fù)制的途徑,但也可成為HBV發(fā)生免疫逃避的作用靶點(diǎn)[2]。TLR10對(duì)TLR2信號(hào)通路下游的炎癥因子具有負(fù)性調(diào)節(jié)作用,有利于HBV發(fā)生免疫逃逸。本研究結(jié)果提示Rs4129009與HBV感染相關(guān),但與HBV感染程度無關(guān);基因變異后易出現(xiàn)高病毒載量、HBeAg陽性。但是,TLR10的負(fù)性調(diào)節(jié)是否通過上述基因介導(dǎo)的信號(hào)通路參與TLR2對(duì)HBV感染的調(diào)控而最終導(dǎo)致HBV慢性感染,尚需進(jìn)一步研究。
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Association of TLR10 Rs4129009 gene polymorphism with chronic hepatitis B virus infection
TANG Yongzhi,CHEN Qiaoling, JIANG Ting,et al.Department of Infectious Diseases,Taizhou Hospital Affiliated to Wenzhou Medical University,Taizhou 317000, China
ObjectiveTo investigate the relationship between the gene polymorphism of Toll like receptor 10(TLR10)and chronic HBVinfection.MethodsThe peripheral blood samples from 428 chronic HBVinfected patients and 210 healthy controls were collected.The extracted whole genome DNA was specifically amplified by polymerase chain reaction(PCR).Then,direct sequencing was performed.ResultsThe characteristics of two groups were in accordence with Hardy-Weinberg balance (both P>0.05).The frequency of AG+GG genotypes and G allele in HBVinfected group were lower than those in healthy control (P<0.01).The subgroup analysis demonstrated that variant gene in HBVDNA>106IU/ml group and HBeAg positive group were higher than the other groups(P<0.01),nevertheless,no significant difference were found in the subgroups of the severity of HBV infection and family medical history(P>0.05).ConclusionPolimorphism of Rs4129009 in TLR10 may be related to chronic HBV infection.
Toll like receptor 10PolymorphismHepatitis B virus
2016-08-25)
(本文編輯:陳丹)
10.12056/j.issn.1006-2785.2017.39.13.2016-1352
臺(tái)州市科技計(jì)劃項(xiàng)目(20111ky0604)
317000溫州醫(yī)科大學(xué)附屬臺(tái)州醫(yī)院感染科(湯永志、陳巧玲、燕飛、陳華忠、朱堅(jiān)勝),科教部(朱敏);臨海市第一人民醫(yī)院感染科(江挺)
朱堅(jiān)勝,E-mail:zhujs@enzemed.com