【作者】 张小花；

【导师】 汤华；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 目的探讨体外培养条件下HBV与DNAJB4基因转录水平表达的关系,揭示HBV诱发肝癌发生的新机制,为HBV感染的治疗提供新的思路,为深入理解DNAJB4的转录调节机制提供新的知识。方法1.用RT-PCR和Real-time PCR检测DNAJB4在HepG2细胞和HepG2.2.15细胞中转录水平的表达。2.构建DNAJB4启动子虫荧光素酶报告质粒,分别与HBV表达质粒、对照质粒共转染HepG2细胞,利用双荧光素酶报告基因检测系统,检测荧光素酶活性。3.构建HBs、HBp、HBc及HBx表达质粒,分别与DNAJB4启动子虫荧光素酶报告质粒共转染HepG2细胞,利用双荧光素酶报告基因检测系统,检测荧光素酶活性。结果1. RT-PCR结果显示DNAJB4在HepG2.2.15细胞中的表达量是其在HepG2细胞中表达量的3倍,Real-time PCR结果显示DNAJB4在HepG2.2.15细胞中的表达量是其在HepG2细胞中表达量的2.59倍。2.成功构建了DNAJB4启动子虫荧光素酶报告质粒,且DNAJB4启动子虫荧光素酶报告质粒与HBV表达质粒共转染组的相对荧光素酶活性是其对照组的2.28倍。3.转染HBs和HBc表达质粒组的相对荧光素酶活性分别是其对照组的2.11倍和1.77倍,而HBx、HBp表达质粒对荧光素酶活性没有影响。结论在HepG2.2.15细胞中,HBV能通过增强DNAJB4启动子活性增加DNAJB4转录水平的表达,其中HBs和HBc蛋白起主要作用。本研究为深入理解DNAJB4的转录调节机制提供了新的知识,为HBV引起的肝癌提供了新的见解。更多还原

【Abstract】 ObjectiveTo explore the relationship between HBV and transcriptional level expression of DNAJB4 in vitro, to reveal new mechanisms of HBV-induced hepatocellular carcinoma, to provide new ideas for the treatment of HBV infection and new knowledge for in-depth understanding of the transcriptional regulation of DNAJB4.Methods1. RT-PCR and Real-time PCR were performed to detect DNAJB4 expression on mRNA level in HepG2 and HepG2.2.15 cell lines.2. DNAJB4 promoter luciferase reporter plasmid, pGL3-Basic-DNAJB4-P, was constructed. HepG2 cells were transiently cotransfected with the same amount of pGL3-Basic-DNAJB4-P and HBV expression plasmids or control plasmids, respectively. Then their luciferase activities were detected.3. HBs, HBp, HBc and HBx expression plasmids were constructed. HepG2 cells were transiently cotransfected with the same amount of pGL3-Basic-DNAJB4-P and HBx or HBs or HBc or HBp expression plasmids, respectively. Then their luciferase activities were detected.Results1. RT-PCR results showed that the amount of DNAJB4 expression on mRNA level in HepG2.2.15 cells is 3 times higher than that in HepG2 cells. Real-time PCR showed that the amount of DNAJB4 expression on mRNA level in HepG2.2.15 cells is 2.59 times higher than that in HepG2 cells. 2. DNAJB4 promoter luciferase reporter plasmid, pGL3-Basic-DNAJB4-P, was successfully constructed. The relative luciferase activity in HepG2 cells, which were transiently cotransfected with pGL3-Basic-DNAJB4-P and HBV expression plasmid, is 2.28 times higher than that in control group.3. The relative luciferase activities in HepG2 cells transfected with HBs or HBc expression plasmids are 2.11 times and 1.77 times higher than those in control group, respectively. However, HBx and HBp expression plasmids had no effect on luciferase activity.ConclusionWe found that HBV upregulates DNAJB4 transcriptional level expression in HepG2.2.15 cells through enhancing its promoter activity, and HBs, HBc proteins may play important roles in enhancing DNAJB4 promoter activity. This study can provide new knowledge for in-depth understanding of the transcriptional regulation mechanism of DNAJB4 and new insights for HBV-induced hepatocellular carcinoma.更多还原