HBV前S2蛋白对胰岛素受体基因下调作用的研究  

Down-regulation effect of hepatitis B virus pre-S2 protein on insulin receptor gene expression

作　　者：苏何玲[1] 纪冬[1] 韩萍[2] 刘妍[1] 张健[2] 陈国凤[2] 钟彦伟[1] 徐东平[1] 

机构地区：[1]解放军第302医院全军传染病研究所病毒性肝炎研究室,北京100039 [2]解放军第302医院全军传染病研究所感染7科,北京100039

出　　处：《解放军医学杂志》2009年第6期679-682,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(30700713)

摘　　要：目的探讨乙型肝炎病毒前S2蛋白(pre-S2)对胰岛素受体(INSR)基因的转录调节作用。方法以pGEM-Teasy-65-2质粒为模板,扩增乙型肝炎病毒(HBV)pre-S2基因片段,构建真核表达载体pcDNA3.1-preS2,以不同剂量(1.0、1.5、2.0μg)转染肝癌细胞系HepG2和Huh7,36h及72h后分别提取转染后细胞总RNA,利用实时荧光定量PCR检测细胞内的INSR mRNA水平,观察HBV的pre-S2蛋白对INSR基因的转录调节作用,同时观察阻断pre-S2蛋白后INSR的表达情况。结果转染不同剂量pcDNA3.1-preS2后,HepG2细胞内INSR基因的表达水平分别为对照的61%、20%、11%(转染后36h)和92%、69%、37%(转染后72h),Huh7细胞内INSR基因的表达水平分别为对照的63%、47%、35%(转染后36h)和83%、73%、34%(转染后72h);细胞INSR mRNA水平随真核表达载体pcDNA3.1-preS2转染剂量的增加而降低。在转染2μgpcDNA3.1-preS2的同时,将抗pre-S2单克隆抗体加入培养上清,可部分阻断pre-S2蛋白对INSRmRNA表达的抑制,HepG2细胞中的INSRmRNA水平分别恢复到转染空质粒对照的65%(转染后36h)和81%(转染后72h),Huh7细胞中的INSRmRNA水平则分别恢复到69%(转染后36h)和96%(转染后72h)。结论HBVpre-S2蛋白对肝癌细胞系HepG2和Huh7细胞的INSR基因有明显下调作用,抗pre-S2抗体能部分阻断这种下调作用,此机制可以在分子水平部分解释肝源性糖尿病的发生。Objective To investigate the regulatory effect of HBV pre-S2 protein on insulin receptor gene expression. Methods The HBV pre-S2 gene was amplified by PCR with plasmid pGEM-Teasy-65-2 as template. The pcDNA3. 1-preS2 was constructed with routine molecular methods and transfected into HepG2 and Huh7 cell lines in different doses （1.0, 1.5, 2.0μg）. Thirty-six and 72 hours after transfection, total RNA was extracted from transfected cells with Trizol reagent, and relative quantification of insulin receptor mRNA was preformed by real-time fluorescent reverse transcription PCR. The regulatory effect of HBV pre-S2 protein on insulin receptor gene expression was investigated, as well as the INSR expression before and after the pre-S2 protein was blocked. Results After transfection, the mRNA levels of insulin receptor gene were 61%, 20%, 11% （36h） and 92%, 69%, 37% （72h） of the controls in HepG2, respectively, and were 63%, 47%, 35% （36h） and 83%, 73%, 34% （72h） of the controls in HuhT. The mRNA levels of insulin receptor gene decreased with the increase of transfection quantity of the pcDNA3. 1-preS2. When the pre-S2 monoclonal antibody was added into the supernatant of transfected HepG2 or Huh7 cell lines, the mRNA levels of insulin receptor gene were partly restored to 65% （36h） and 81% （72h） of the controls in HepG2, and 69% （36h） and 96% （72h） in Huh7. Conclusion The insulin receptor gene can be down-regulated by hepatitis B virus pre-S2 protein in HepG2 and Huh7 cell lines, and the result can partly elucidate the pathogenesis of hepatic diabetes at molecular level.

关 键 词：肝炎病毒 乙型 前S2蛋白 受体 胰岛素 基因调节 

分 类 号：R512.63[医药卫生—内科学]