王倩，吳鵬波，李明，羅和生，余媛潔，譚詩(shī)云，舒泳翔(武漢大學(xué)人民醫(yī)院，武漢430060)

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VEGF基因多態(tài)性及吸煙在誘發(fā)非酒精性脂肪肝中的交互作用

王倩，吳鵬波，李明，羅和生，余媛潔，譚詩(shī)云，舒泳翔(武漢大學(xué)人民醫(yī)院，武漢430060)

摘要：目的觀察非酒精性脂肪肝(NAFLD)患者血管內(nèi)皮生長(zhǎng)因子(VEGF)基因rs833061、rs3025039位點(diǎn)多態(tài)性，并探討其多態(tài)性及吸煙在誘發(fā)NAFLD中的交互作用。方法選擇NAFLD患者341例(NAFLD組)、健康體檢者246例(對(duì)照組)，采用PCR-RFLP法分析VEGF基因rs833061、rs3025039位點(diǎn)基因型，非條件Logistic回歸分析法分析各基因型與NAFLD易感性的關(guān)系，非條件Logistic回歸分析法和廣義多因子降維法分析VEGF基因多態(tài)性及吸煙在誘發(fā)NAFLD中的交互作用。結(jié)果VEGF基因rs833061位點(diǎn)C基因攜帶者(CC+CT)罹患NAFLD的風(fēng)險(xiǎn)較非C基因攜帶者(TT)升高(P均<0.01)，rs3025039位點(diǎn)T基因攜帶者(TT+CT)患病風(fēng)險(xiǎn)較非T基因攜帶者(CC)風(fēng)險(xiǎn)升高(P均<0.01)。VEGF基因rs833061位點(diǎn)多態(tài)性與吸煙可能在 NAFLD發(fā)病過(guò)程中存在相加作用(P<0.01)，rs833061位點(diǎn)CC/CT基因型吸煙個(gè)體罹患NAFLD風(fēng)險(xiǎn)是攜帶TT基因型非吸煙個(gè)體的4.93倍。結(jié)論VEGF基因多態(tài)性與NAFLD發(fā)病有關(guān)，VEGF基因rs833061位點(diǎn)多態(tài)性與吸煙在誘發(fā)NAFLD過(guò)程中具有協(xié)同效應(yīng)。

關(guān)鍵詞：非酒精性脂肪肝；血管內(nèi)皮生長(zhǎng)因子；基因多態(tài)性；易感性；交互作用

非酒精性脂肪肝(NAFLD)不僅可進(jìn)展為肝硬化甚至肝癌，同時(shí)還可增加高血壓、糖尿病等的罹患風(fēng)險(xiǎn)[1,2]?，F(xiàn)已明確，遺傳因素在NAFLD發(fā)病過(guò)程中具有重要作用[3]。血管內(nèi)皮生長(zhǎng)因子(VEGF)可促進(jìn)血管內(nèi)皮細(xì)胞增殖、分化，增加血管通透性。此外，VEGF還具有調(diào)節(jié)能量代謝、胰島素抵抗以及炎癥反應(yīng)等生物學(xué)效應(yīng)[4,5]。研究發(fā)現(xiàn)，吸煙不僅加劇胰島素抵抗，還可導(dǎo)致向心性肥胖[6]。而胰島素抵抗及向心性肥胖是NAFLD發(fā)病的重要危險(xiǎn)因素。本研究探討NAFLD患者VEGF基因rs833061、rs3025039位點(diǎn)多態(tài)性及吸煙在誘發(fā)NAFLD中的交互作用。

1資料與方法

1.1臨床資料選擇2012年2月～2014年8月本院收治的NAFLD患者341例(NAFLD組)，診斷標(biāo)準(zhǔn)符合《非酒精性脂肪性肝病診療指南(2010年修訂版)》[7]。其中，男210例、女131例，年齡(46.69±9.87)歲。同期另選在該院體檢健康者246例(對(duì)照組)，男170例、女76例，年齡(47.65±11.36)歲。兩組基本資料比較見(jiàn)表1。

表1　兩組基本資料比較

注：HOMA-IR為胰島素抵抗指數(shù)；與對(duì)照組比較，*P<0.05，△P<0.01。

1.2VEGF基因rs833061、rs3025039位點(diǎn)多態(tài)性檢測(cè)采用PCR-RFLP法。所有研究對(duì)象抽取空腹靜脈血，采用酚-氯仿法提取基因組DNA。根據(jù)VEGF-9及VEGF-2基因的核苷酸序列設(shè)計(jì)引物，采用PCR擴(kuò)增目的基因片段。VEGF基因rs833061位點(diǎn)引物序列：上游引物：5′-CTCTTTAGCCAGAGCCGGGG-3′，下游引物：5′-TGGCCTTCTCCCCGCTCCGAC-3′；VEGF基因rs3025039位點(diǎn)引物序列：上游引物5′-AGGGTTCGGGAACCAGATC-3′，下游引物5′-CTCGGTGATTTAGCAGCAAG-3′。PCR反應(yīng)體系25 μL，包括DNA 100 ng、1.0 U Taq酶、2 μL上下游引物、2×PCR buffer；PCR反應(yīng)條件：94 ℃ 5 min，94 ℃ 30 s、60 ℃ 30 s、72 ℃ 45 s，30個(gè)循環(huán)，最后72 ℃延伸10 min。限制性內(nèi)切酶BsaHⅠ、NlaⅢ酶切。酶切產(chǎn)物經(jīng)3%瓊脂糖凝膠電泳，EB染色成像，判定酶切基因型。

2結(jié)果

2.1VEGF基因rs833061、rs3025039位點(diǎn)基因型VEGF基因rs833061位點(diǎn)TT基因型片段長(zhǎng)度為230、75 bp(75 bp片段太小，跑出膠外)，CT基因型片段長(zhǎng)度為290、230 bp，CC基因型片段長(zhǎng)度為290 bp。VEGF基因rs3025039位點(diǎn)TT基因型片段長(zhǎng)度為195 bp(25 bp片段太小，跑出膠外)，CC基因型片段長(zhǎng)度為195、170 bp，CT基因型片段長(zhǎng)度為170 bp。

2.2兩組VEGF基因rs833061、rs3025039位點(diǎn)基因型分布及其與NAFLD易感性的關(guān)系兩組VEGF基因rs833061、rs3025039位點(diǎn)基因型分布見(jiàn)表2。相關(guān)分析顯示，VEGF基因rs833061位點(diǎn)C基因攜帶者(CC+CT)罹患NAFLD的風(fēng)險(xiǎn)是非C基因攜帶者(TT)的1.56倍(95%CI為1.12～2.15，P<0.05)；校正吸煙、腰圍、血糖等因素后，C基因攜帶者(CC+CT)是非C基因攜帶者(TT)的1.71倍(95%CI為1.42～2.22，P<0.01)。VEGF基因rs3025039位點(diǎn)T基因攜帶者(TT+CT)罹患NAFLD的風(fēng)險(xiǎn)是非T基因攜帶者(CC)的1.61倍(95%CI為1.15～2.26，P<0.05)；校正吸煙、腰圍、血糖等因素后，T基因攜帶者(TT+CT)罹患NAFLD的風(fēng)險(xiǎn)是非T基因攜帶者(CC)的1.83倍(95%CI為1.35～2.71，P<0.01)。

表2　兩組VEGF基因rs833061、rs3025039

2.3VEGF基因rs833061、rs3025039位點(diǎn)多態(tài)性與吸煙對(duì)誘發(fā)NAFLD的交互作用VEGF基因rs833061位點(diǎn)多態(tài)性+吸煙模型具有最大的預(yù)測(cè)正確率(0.668 8)和最高的交叉驗(yàn)證一致性(10/10)，為最優(yōu)模型。說(shuō)明二者存在交互作用。與攜帶VEGF基因rs833061位點(diǎn)TT基因型不吸煙者相比，攜帶VEGF基因rs833061位點(diǎn)CT/CC基因型不吸煙者罹患NAFLD的風(fēng)險(xiǎn)增加2.22倍(P<0.01)，攜帶VEGF基因rs833061位點(diǎn)TT基因型吸煙者罹患NAFLD風(fēng)險(xiǎn)增加1.82倍(P<0.01)，攜帶VEGF基因rs833061位點(diǎn)CT/CC基因型吸煙者罹患NAFLD風(fēng)險(xiǎn)增加4.93倍(P<0.01) ；VEGF基因rs833061位點(diǎn)多態(tài)性與吸煙對(duì)誘發(fā)NAFLD存在相加作用(P<0.01)。

3討論

近年研究證實(shí)，遺傳因素在NAFLD發(fā)病中發(fā)揮重要作用[3]。人類(lèi)VEGF基因位于染色體6p21.3，其蛋白表達(dá)水平在不同個(gè)體間有一定差異，這可能與VEGF基因多態(tài)性有關(guān)[9]。VEGF基因多態(tài)性位點(diǎn)主要位于5′非翻譯區(qū)和3′非翻譯區(qū)。研究發(fā)現(xiàn)，VEGF基因多態(tài)性與肥胖、多囊卵巢綜合癥易感性相關(guān)[10,11]，而肥胖及多囊卵巢綜合癥與脂肪肝在易感基因方面存在一定相似性[12,13]。因此，VEGF基因多態(tài)性可能與NAFLD發(fā)病有關(guān)。

本文通過(guò)病例對(duì)照分析發(fā)現(xiàn)，以TT為參照，VEGF基因rs833061位點(diǎn)攜帶CT、CC基因型個(gè)體罹患NAFLD的風(fēng)險(xiǎn)增加；以CC為參照，VEGF基因rs3025039位點(diǎn)攜帶CT、TT基因型個(gè)體罹患NAFLD的風(fēng)險(xiǎn)增加。Logistic回歸校正腰圍、血糖、HDL-C、HOMA-IR及吸煙等因素后，仍發(fā)現(xiàn)rs833061、rs3025039位點(diǎn)多態(tài)性對(duì)NAFLD易感性有顯著影響。

NAFLD發(fā)病是個(gè)體遺傳因素與外界環(huán)境因素共同作用的結(jié)果。吸煙是NAFLD發(fā)生的危險(xiǎn)因素，吸煙個(gè)體罹患NAFLD的風(fēng)險(xiǎn)增加25%[11]。本研究采用GMDR法分析VEGF基因rs833061、rs3025039位點(diǎn)多態(tài)性與吸煙對(duì)誘發(fā)NAFLD的交互作用。GMDR法是研究基因-基因以及基因-環(huán)境交互作用的一種可靠方法[12]。結(jié)果提示，rs833061位點(diǎn)多態(tài)性和吸煙在NAFLD發(fā)病中存在交互作用，而rs3025039位點(diǎn)多態(tài)性與吸煙則無(wú)交互作用。進(jìn)一步采用非條件Logistic回歸分析法及Bootstrap法[14]對(duì)上述結(jié)果加以驗(yàn)證，結(jié)果提示rs833061位點(diǎn)多態(tài)性和吸煙存在相加作用，但二者相加作用的機(jī)制目前尚不清楚。Fesinmeyer等[15]研究認(rèn)為，吸煙可能是通過(guò)改變r(jià)s833061位點(diǎn)多態(tài)性協(xié)同增加NAFLD易感性。

綜上所述， VEGF基因rs833061、rs3025039位點(diǎn)多態(tài)性與NAFLD發(fā)病有關(guān)，VEGF基因rs833061位點(diǎn)多態(tài)性與吸煙在誘發(fā)NAFLD過(guò)程中具有相加作用，但上述結(jié)果仍需擴(kuò)大樣本量進(jìn)一步證實(shí)。

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Gene polymorphism of VEGF and its interaction with smoking in patients with non-alcoholic fatty liver disease

WANGQian,WUPengbo,LIMing,LUOHesheng,YUYuanjie,TANShiyun,SHUYongxiang

(RenminHospitalofWuhanUniversity,Wuhan430060,China)

Abstract:ObjectiveTo observe the gene polymorphisms of vascular endothelial growth factor (VEGF) rs833061 and rs3025039 and to investigate their interaction with smoking in patients with non-alcoholic fatty liver disease (NAFLD). MethodsGenotypes of VEGF rs833061 and rs3025039 in 341 patients with NAFLD (NAFLD group) and 246 control subjects (control group) were examined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). The unconditional logistic regression (ULR) was performed to analyze the relationships between the genotypes and susceptibility to NAFLD. The interactions between genotypes of VEGF rs833061 and rs3025039 and smoking in inducing NAFLD were explored by generalized multifactor dimensionality reduction (GMDR) and unconditional Logistic regression method. ResultsThe risk of NAFLD in VEGF rs833061 with genotype C (CC+CT) was higher than that of carriers without C (TT) (all P<0.01), and the risk of NAFLD in rs3025039T gene carriers (TT+CT) was higher than that of carriers without genotype T (CC) (all P<0.01). Gene polymorphism of VEGF rs833061 had interactions with smoking in the process of NAFLD (P<0.01). The risk of NAFLD in rs833061 CC/CT genotype of smoking individuals was 4.93 times higher than that of TT genotype of non-smoking individuals. ConclusionsVEGF gene polymorphism is associated with the risk of NAFLD. VEGF rs833061 and smoking have synergistic effects in the process of inducing NAFLD.

Key words:non-alcoholic fatty liver disease; vascular endothelial growth factor; gene polymorphisms; susceptibility; interaction

(收稿日期：2015-07-24)

中圖分類(lèi)號(hào)：R575.5

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1002-266X(2016)08-0007-03

doi:10.3969/j.issn.1002-266X.2016.08.003

通信作者簡(jiǎn)介：譚詩(shī)云(1962-)，男，主任醫(yī)生，教授，研究方向?yàn)楦闻K相關(guān)疾病的基礎(chǔ)與臨床。E-mail： tanshiyun@126.com

作者簡(jiǎn)介：第一王倩(1989-)，女，碩士在讀，研究方向?yàn)榉蔷凭灾靖蔚脑\治。E-mail： 1028578861@qq.com

基金項(xiàng)目：湖北省自然科學(xué)基金重點(diǎn)項(xiàng)目(2014CFA045)。