论文部分内容阅读
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.
OBJECTIVE: To establish an animal model of transgenic mice that consistently expresses and induces expression of the structural gene of hepatitis C virus (HCV), including the core and envelope proteins E1 and E2, and to explore the pathology of hepatic steatosis induced by HCV structural gene expression (DNA fragment of about 2.2kb) was amplified by polymerase chain reaction (PCR) and cloned into eukaryotic expression vector pcDNA4HisMas and pMT / BiP / V5 -HisA were constructed into the expression vector pcDNA4HisMas-HCVCore-E2 and the expression vector pMT / BiP / V5-HisA-HCVCore-E2 respectively.The expression of restriction endonuclease and the nucleotide sequence of the target DNA fragment Sequence analysis confirmed that the constructed eukaryotic expression vector of HCV structural gene was correct.Transfection of COS-7 cell line with liposome in vitro and Western blot analysis using polyclonal anti-HCV E2 antibody confirmed that transfected cells have HCVE2 protein .In this study, the DNA of the linearized expression vector was introduced into the nucleus by microinjection of mouse fertilized eggs and the transgenic mouse model expressing the HCV structural gene was established by fertilized egg implantation in pseudopregnant mice. And the liver tissues of the dead mice, 6-month-old and copper sulfate induced expression transgenic mouse models of continuous expression transgenic mice were analyzed by HE staining and pathological characteristics.Results: Consistent expression and inducible expression HCV transfected cells were transfected with pcDNA4HisMas-HCVCore-E2 and pMT / BiP / V5-HisA-HCVCore-E2, and the expression of HCVE2 antigen in transfected cells was confirmed by transfection of COS-7 cells in vitro. Liver pathological features were analyzed and found that the liver has not yet completed the development of differentiation, a large number of hematopoietic cells in the liver, hepatocyte vacuolar degeneration, mononuclear cell aggregation phenomenon for sustained expression of HCV structural gene 6-month-old transgenic mice liver Pathological analysis found that the central venous area of hepatocyte vesicle-induced steatosis, central vein around the cell lysis and necrosis, central venous was significantly expanded, a small number of organizations also see bubble-shaped steatosis.Induced transgenic mouse liver disease Neutrophil analysis found that the hepatic lobule within the central venous surrounding the necrosis involving 5-10 lobular lymphocytes around the infiltration, the junction area can be seen in most of the liver cell nucleus Often, or solidified, or garland, or dissolved into a light blue amorphous matrix.A large number of fat vesicles left in the pulp slurry, liver steatosis and nuclear abnormalities have a migration process.Conclusion: The establishment of the HCV gene expression gene transgene Mice and found that HCV structural gene in liver of transgenic mice has a typical steatosis pathological changes for the study of HCV infection caused by chronic hepatitis C combined with steatosis characteristics and mechanism of the study laid the foundation.