HBV感染诱发AID高表达与肝细胞癌变相关性研究  

Relation between hepatocellular carcinogenesis and an aberrant expression of activation induced deaminase triggered by chronic HBV infection

作　　者：乔莹[1] 黄芬[2] 蔡鑫泽[1] 阚周密[1] 赵连爽[1] 管格非[1] 梁国新[1] 

机构地区：[1]中国医科大学附属第一医院中心实验室,辽宁沈阳110001 [2]中国医科大学附属盛京医院感染科,辽宁沈阳110001

出　　处：《中华肿瘤防治杂志》2015年第1期13-18,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：教育部留学归国启动基金(教外司2009-8);辽宁省自然科学基金(200904409)

摘　　要：目的通过研究乙型肝炎病毒(hepatitis B virus,HBV)长期感染与诱导性脱氨酶(activation induced deaminase,AID)表达水平的关系探讨肝癌发病机制。方法采用实时定量PCR法,测定乙肝病毒感染及转化生长因子β1(TGF-β1)和肿瘤坏死因子-α(TNF-α)对Apobec蛋白家族AID和Apobec3G表达量的影响;采用3D-PCR法检测AID、Apobec3G表达组对乙肝病毒基因突变的影响;克隆测序PCR扩增乙肝病毒X基因产物,对比AID和Apobec3G对乙肝病毒基因组突变的影响,计算突变率;并用逆转录病毒携带AID组和对照组在肝脏细胞转染表达后,克隆测序PCR细胞部分基因扩增,对比测定对细胞基因组突变的影响,计算突变率。结果乙肝病毒感染或生长因子TGF-β1刺激使肝细胞中AID表达上调>10倍,TGFβ1刺激组较非刺激组引起乙肝病毒基因组高突变,分别为55个克隆中的16和4个。AID高表达使乙肝病毒基因组高度突变,突变率为55个克隆中的16个,显著高于对照组的1个,多为G到A突变;而且AID高表达使部分细胞基因组如p53和c-myc基因高度突变,突变率分别为8.2×10-5和7.3×10-5,高于对照组的突变率0。结论慢性肝炎病毒感染协同细胞因子诱导AID高表达,高表达的AID使肝炎病毒基因及细胞基因组突变,为肝细胞癌变的相关因素。OBJECTIVE By studying the relationship between chronic hepatitis B virus infection and expression levels of activatiowinduced cytidine deaminase, to investigate the potential mechanism inducing hepatocellular carcinogenesis. METHODS By employing Real-time PCR, we analyzed the expression of Apobec family AID and Apobec3G induced by HBV infection and cytokine TGF-β and TNF-α stimulation. By performing 3D-PCR we analyzed the effect of AID and Apobec3G on HBV hypermutations. The X gene mutation frequency of HBV virus was obtained by cloning and sequen- cing. Compared and calculated the AID and Apobec3G ability to induce HBV genome hypermuation. Also, by utilizing retrovirus to express AID and Mock in hepatocyte cells and performing PCR to amplify several cell genes for cloning and sequencing, the cellular gene mutation frequencies were obtained and compared. RESULTS HBV infection and transfor mation growth factor TGFβ1 promoted cell to upregulate AID expression over 10-fold. Meanwhile, TGFβ1 could induce more hypermuation on HBV virus genome than the control group：the form was found to have 16 mutations in 55 clones and latter was 4 mutations. The high AID expression induced not only HBV genome hypermutation （inducing 16 mutations in 55 clones）, but also several cell gene mutations, including p53 and c-myc mutations. The mutation frequencies were 8.2 ×10-5 and 7. 3×10-5 respectively, which was much higher than the control group mutation frequency 0.CONCLUSIONS Chronic HBV infection and cytokine synergically incudces high expression of All）; high expression of AID induces the mutation of both HBV and human genome, which is considered as one of potential factors to accelerate hepatocellular cancer progression.

关 键 词：肝肿瘤 乙型肝炎病毒 诱导性脱氨酶 转化生长因子β1 基因超突变 

分 类 号：R735.7[医药卫生—肿瘤]