丙型肝炎病毒核心蛋白对基质金属蛋白酶组织抑制因子-1表达的影响

发布时间：2018-06-12 20:35

本文选题：丙型肝炎病毒核心蛋白 + 基质金属蛋白酶组织抑制因子-1启动子　；参考：《兰州大学》2006年硕士论文

【摘要】：目的肝硬化是丙型肝炎严重的并发症之一,然而丙型肝炎病毒(HCV)引起纤维化、肝硬化的机制尚不清楚,鉴于大量文献报道HCV核心蛋白在细胞生长及肝癌发生方面具有重要调控作用,我们采用分子生物学方法研究HCV核心蛋白对TIMP-1基因表达的影响,以探讨HCV的致肝纤维化机制。方法 1.以人基因组DNA为模板,根据参考文献设计引物,采用聚合酶链反应,克隆TIMP1p。构建TIMP1p的氯霉素乙酰转移酶(CAT)报告基因表达载体转染HepG2细胞,同时平行设立Pcat3-basic阴性对照组、Pcat3-control阳性对照组,用ELISA法检测各组氯霉素乙酰转移酶(CAT)的表达来鉴定TIMP1启动子的活性。 2.应用瞬时转染技术,将PcDNA3.1(-)-HCVcore与Pcat3-TIMP-1p共转染入LX-2细胞,同时平行设立Pcat3-basic阴性对照组、Pcat3-control阳性对照组、Pcat3-TIMP-1p单独转染组,用ELISA法检测各组CAT的表达来鉴定HCV核心蛋白对TIMP1表达的影响。结果 1.成功克隆TIMP1启动子片断,经测序鉴定正确。成功构建了TIMP1p的CAT报告基因表达载体,Pcat3-TIMP-1p转染HepG2细胞后,EHSA法检测结果显示Pcat3-basic的吸光度值为0.004±0.002,Pcat3-TIMP-1p为2.329±0.685,经方差分析各组差异有统计学意义,F=26.075(P0.05),LSD法两两比较均显示P0.05,证明TIMP-1p能够启动CAT的表达。 2.PcDNA3.1(-)-HCVcore与Pcat3-TIMP-1p共转染LX-2细胞后,ELISA法检测结果显示Pcat3-TIMP-1p吸光度值为0.833±0.040,同期单独转染LX-2细胞的Pcat3-TIMP-1p吸光度值为0.677±0.049,方差分析各组差异有统计学意义,F=132.401(P0.05),LSD法两两比较均显示P0.05,提示PcDNA3.1(-)-HCVcore与Pcat3-TIMP-1p共转
[Abstract]:Objective liver cirrhosis is one of the serious complications of hepatitis C. However, the mechanism of cirrhosis is not clear because of the fibrosis caused by hepatitis C virus (HCV). As a large number of literatures have reported that HCV core protein plays an important role in the regulation of cell growth and hepatocellular carcinogenesis, we used molecular biological methods to study the effect of HCV core protein on TIMP-1 gene expression in order to explore the mechanism of hepatic fibrosis induced by HCV. Method 1. Using human genomic DNA as template, primers were designed according to references, and TIMP 1p was cloned by polymerase chain reaction (PCR). The expression vector of chloramphenicol acetyltransferase (CAT) reporter gene of TIMP1p was constructed and transfected into HepG2 cells, and the Pcat3-basic negative control group was set up in parallel with Pcat3-control positive control group. Enzyme linked immunosorbent assay (Elisa) was used to detect the expression of chloramphenicol acetyltransferase (CAT) in order to identify the activity of TIMP1 promoter. Using transient transfection technique, PcDNA3.1 ~ (-1) -Pcat3-TIMP-1p was co-transfected into LX-2 cells, and Pcat3-basic negative control group (Pcat3-control) was established in parallel. The expression of cat in each group was detected by Elisa to determine the effect of HCV core protein on the expression of TIMP _ (1) in LX-2 cells, and Pcat3-TIMP-1p was transfected into LX-2 cells in parallel with Pcat3-basic negative control group. The expression of cat in each group was detected by Elisa. Result 1. The promoter of TIMP1 was successfully cloned and confirmed by sequencing. The cat reporter gene expression vector of TIMP1p was successfully constructed. After transfected HepG2 cells with Pcat3-TIMP-1p, the results of EHSA assay showed that the absorbance of Pcat3-basic was 0.004 卤0.002, Pcat3-TIMP-1p was 2.329 卤0.685, and the difference of F26.075P0.05LSD between the two groups was statistically significant by ANOVA analysis of variance, which proved that TIMP-1p could be detected in both groups. 2. After PcDNA3.1 ~ + -HCVcore and Pcat3-TIMP-1p co-transfected LX-2 cells, the results of Elisa showed that the absorbance of Pcat3-TIMP-1p was 0.833 卤0.040, and the absorbance of Pcat3-TIMP-1p was 0.677 卤0.049 when LX-2 cells were transfected alone at the same time. There was statistical significance in the analysis of the differences among the three groups. There was significant difference between the two groups (P < 0.05), indicating that the PcDNA3.1 ~ (-1) -ti-HCV core and Pcat3-TIMP-1p were cotransferred with Pcat3-TIMP-1p.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373.21

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