丙型肝炎病毒感染与白细胞介素27的相关性研究

发布时间：2018-01-13 11:45

本文关键词：丙型肝炎病毒感染与白细胞介素27的相关性研究　出处：《武汉大学》2011年博士论文　论文类型：学位论文

【摘要】：人类丙型肝炎的病原体是丙型肝炎病毒(hepatitis C virus, HCV)。世界上1.8亿人感染HCV,感染率约为3%,但50%～80%的病人会变成慢性感染者,其中部分慢性感染者可发展为肝纤维化、肝硬化,或者肝细胞癌。已有的研究表明,白细胞介素在HCV感染过程中起到重要作用。白细胞介素27(interleukin27, IL-27)是IL-6/IL-12家族的新成员,是一个异源二聚体,由EBI3和P28两个亚基组成,其中EBI3与IL-12的p40亚基同源,p28与IL-12的p35亚基同源。近年来有研究发现IL-27能够抑制HIV在PBMC细胞,CD4+T细胞,巨噬细胞中的复制。2009年有报道IL-27融合蛋白对肝细胞和肝癌细胞有类似IFN-γ的作用,认为IL-27很可能具有在肝细胞和肝癌细胞中抗病毒活性,并发现IL-27可以抑制禽流感病毒和人流感病毒在HepG2细胞中的复制。因此,IL-27在宿主抗病毒感染过程中可能有重要作用。但到目前为止,关于HCV感染与IL-27的关系尚不明确。为了探讨HCV感染与IL-27的关系,我们检测了慢性丙肝患者和健康对照者血清中IL-27的蛋白水平。临床数据表明,慢性丙肝患者血清中IL-27的蛋白水平显著升高。此后,我们在体外细胞试验中同样证明了HCV的感染可以增强IL-27的表达水平,并初步探讨了HCV上调IL-27表达水平的分子机制。在IL-27p28亚基和EBI3亚基的启动子水平,我们通过报告基因检测系统,发现HCV能分别上调p28亚基和EBI3亚基启动子的活性。我们用RT-PCR和ELISA检测了HCV感染对IL-27的转录水平和蛋白表达水平的影响,发现HCV感染显著上调了IL-27p28亚基和EBI3亚基的mRNA和蛋白表达量。通过对信号通路的研究,我们进一步发现HCV株JFH-1可以通过NF-κB通路激活IL-27亚基EBI3启动子,而HCV NS3蛋白通过激活NF-κB通路上调IL-27EBI3亚基启动子活性。最后,我们还探讨IL-27对于HCV复制的影响。我们采用HCV病毒株JFH-1感染Huh7.5.1细胞以产生HCV颗粒,用IL-27融合蛋白刺激感染后细胞,并用荧光定量PCR法检测感染细胞培养基中HCV拷贝数,结果发现IL-27融合蛋白在细胞水平能够抑制HCV的复制。该实验结果表明在HCV感染会导致IL-27的表达水平明显提高,而且进一步研究初步揭示了HCV调节IL-27表达的分子机制,最后还提示IL-27可能抑制HCV的复制。为更好地了解HCV病人机体的自然保护机制和研发抗HCV治疗方案提出了新思路。
[Abstract]:Hepatitis C virus is the pathogen of human hepatitis C. 180 million people in the world are infected with hepatitis C virus, and the infection rate is about 3%. But 50 percent of patients become chronically infected, some of whom can develop liver fibrosis, cirrhosis, or hepatocellular carcinoma. Interleukin 27 (IL 27) is a new member of IL-6/IL-12 family. It is a heterodimer composed of two subunits, EBI3 and P28, in which EBI3 is homologous to the p40 subunit of IL-12. P28 is homologous to the p35 subunit of IL-12. In recent years, it has been found that IL-27 can inhibit HIV in PBMC cells. Replication in macrophages. In 2009, it was reported that IL-27 fusion protein had IFN- 纬 similar effects on hepatocytes and hepatoma cells. It is suggested that IL-27 may have antiviral activity in hepatocytes and hepatoma cells, and that IL-27 can inhibit the replication of avian influenza virus and human influenza virus in HepG2 cells. IL-27 may play an important role in the process of host anti-virus infection, but the relationship between HCV infection and IL-27 is unclear. In order to investigate the relationship between HCV infection and IL-27, we measured the serum IL-27 protein levels in chronic hepatitis C patients and healthy controls. The level of IL-27 protein in serum of patients with chronic hepatitis C was significantly increased. Since then, we have also demonstrated in vitro cell tests that HCV infection can enhance the expression of IL-27. At the promoter level of IL-27p28 subunit and EBI3 subunit, we used the reporter gene detection system to study the molecular mechanism of up-regulation of IL-27 expression by HCV. It was found that HCV could up-regulate the promoter activity of p28 subunit and EBI3 subunit, respectively. RT-PCR and ELISA were used to detect the transcriptional level and protein expression level of HCV infection on IL-27. The impact. It was found that HCV infection upregulated the expression of mRNA and protein of IL-27p28 subunit and EBI3 subunit. We further found that HCV JFH-1 can activate EBI3 promoter of IL-27 subunit through NF- 魏 B pathway. HCV NS3 protein upregulated the promoter activity of IL-27EBI3 subunit by activating NF- 魏 B pathway. Finally, we also investigated the effect of IL-27 on HCV replication. We used HCV virus strain JFH-1 to infect Huh7.5.1 cells to produce HCV particles. IL-27 fusion protein was used to stimulate the infected cells and the HCV copy number in the culture medium of infected cells was detected by fluorescence quantitative PCR. The results showed that IL-27 fusion protein could inhibit the replication of HCV at cell level. The results showed that the level of IL-27 expression was significantly increased after HCV infection, and further studies revealed the molecular mechanism of HCV regulating the expression of IL-27. Finally, it is suggested that IL-27 may inhibit the replication of HCV, which provides a new idea for better understanding of the nature protection mechanism of HCV patients and for the development of anti-#en3# therapy.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373.21

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