丙型肝炎病毒结构基因转基因小鼠引起肝脏脂肪变  

Liver steatosis of transgenic mice ex-pressing hepatitis C virus structural proteins

作　　者：成军[1] 任进余[2] 李莉[1] 陆志檬[2] 李克[1] 洪源[1] 陆荫英[1] 王刚[1] 刘妍[1] 张玲霞[1] 陈菊梅[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心,全军病毒性肝炎防治研究重点实验室,北京市100039 [2]上海第二医科大学瑞金医院临床免疫室,上海20004

出　　处：《世界华人消化杂志》2002年第9期1022-1026,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助课题;No.C39970674;No.C03011402;军队回国留学人员启动基金项目;No.98H038~~

摘　　要：目的:建立持续表达和诱导表达型丙型肝炎病毒(HCV)结构基因(包括核心蛋白和包膜蛋白E1、E2编码基因)的转基因小鼠动物模型,探讨HCV结构基因表达导致肝脏脂肪变的病理改变.方法:以含有近全长HCVcDNA的质粒为模板,通过多聚酶链反应(PCR)技术扩增HCV结构基因的DNA片段(约2.2kb),克隆到真核表达载体pcDNA4HisMas和pMT/BiP/V5-HisA中,分别构建成持续表达型表达载体pcDNA4HisMas-HCVCore-E2和诱导表达型表达载体pMT/BiP/V5-HisA-HCVCore-E2.以限制性内切酶分析和目的DNA片段的核苷酸序列分析,证实构建HCV结构基因的真核表达载体正确无误.以脂质体体外转染COS-7细胞系,利用抗-HCVE2的多克隆抗体进行Westernblot杂交分析,证实转染细胞中有HCVE2蛋白的表达.利用小鼠受精卵细胞核微注射方法,导入线性化表达载体的DNA,利用假孕小鼠的受精卵植入方法,建立了表达HCV结构基因的转基因小鼠模型.对于持续表达型转基因小鼠的死胎、6月龄以及硫酸铜诱导表达型的转基因小鼠模型的肝脏组织,进行HE染色和病理学特征的分析.结果:构建了持续性表达和可诱导性表达型的HCV结构基因表达载体pcDNA4HisMas-HCVCore-E2和pMT/BiP/V5-HisA-HCVCore-E2,体外转染COS-7细胞证实转染细胞中有HCVE2抗原的表达.对于持续表达型转基因小鼠死胎肝脏病理学特征进行分析,发现肝组织尚未分化发育完成,肝内有大量造血细胞,肝细胞空泡变性,单个核细胞聚集现象.对于持续表达HCV结构基因的6月龄转基因小鼠的肝脏进行病理学分析,发现中央静脉区肝细胞小泡型脂肪变性,中央静脉周围细胞溶解坏死,中央静脉明显扩大,少数组织中亦见大泡型脂肪变性.对于可诱导型转基因小鼠的肝脏进行病理学分析,发现肝小叶内中央静脉周围坏死区累及5-10个小叶,周围无淋巴细胞浸润,交界区可见多数肝细胞核异常,或固化,或呈花环状,或溶解为淡�AIM: To establish transgenic mice expressing structural pro-teins of hepatitis C virus (HCV), and to elucidate the patho-logical characteristics of the transgenic mice.METHODS:Structural protein coding gene of HCV was am-plified by polymerase chain reaction (PCR) using plasmidcontaining full length HCV cDNA as the template. Stableand expressive vectors pcDNA4HisMas-HCV Core-E2 andpMT/BiP/V5-HisA-HCV Core-E2 were constructed by insert-ing HCV structural protein coding genes into eukaryotic ex-pressive vectors pcDNA4HisMas and pMT/BiP/V5-HisA,respectively. The expression of HCV E2 protein from trans-fected COS-7 cell line was confirmed with Western blot byusing anti-HCV E2 polyclonal antibody. The liver sampleswere collected from dead mouse embryo, living mouse at 6months old, and induced transgenic mice with copper sul-fate solution, respectively. Liver tissue sections were exam-ined for steatosis by H-E staining. RESULTS: Stable expressive vector pcDNA4HisMas-HCVCore-E2 and inducible vector pMT/BiP/V5-HisA-HCV Core-E2 were successfully constructed, respectively. The expres-sion of HCV E2 protein was confirmed by Western blot. Inthe stable transgenic mice, steatosis could be found in bothdead embryo and living mice at 6 months old. In the in-duced mice, the authors found microvescicle type,macrovescicle type and mixed type of steatosis in the livertissue sections.CONCLUSIONS: Steatosis is the typical pathological changesin the transgenic mice expressing HCV structural proteins inboth stable and inducible vector transgenic mice.

关 键 词：动物模型 丙型肝炎病毒 结构基因 转基因 肝脏脂肪变 

分 类 号：R373.21[医药卫生—病原生物学]