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,或溶解为淡蓝色无定形基质.泡浆内残留大量脂肪小泡,肝脏脂肪变与核异常有一移行过程.结论:建立了表达HCV结构基因的转基因小鼠,发现HCV结构基因的转基因小鼠肝脏有典型的脂肪变病理改变,为进一步研究HCV感染引起的慢性丙型肝炎合并脂肪变的特征和机制研究奠定了基础.
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.
出处
《世界华人消化杂志》
CAS
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~~
