主要穹窿蛋白介导宿主抗病毒先天免疫及分子机制

发布时间：2018-07-01 13:48

本文选题：病毒侵染 + 主要穹窿蛋白　；参考：《武汉大学》2012年博士论文

【摘要】：丙型肝炎病毒(HCV)感染成为危害公众健康的主要问题。全世界有超过1.7亿人感染HCV,其中在中国有超过3000万人感染HCV。主要穹窿蛋白(MVP)是穹窿蛋白的一个主要组成部分,它在细胞耐药性、核质运输、细胞信号通路中起到关键作用。目前为止,关于HCV感染与MVP的关系尚不清楚。为了探讨HCV感染与MVP的关系,我们检测了HCV患者与健康人MVP mRNA和蛋白的表达差异。结果表明,和健康人比,HCV患者PBMC细胞中的MVP mRNA表达水平明显升高。同样,在HCV患者血清和肝组织中,MVP蛋白的表达水平也显著升高。因为Huh7细胞系和Huh7.5.1细胞系是一种有效的的HCV感染的细胞培养模型,所以我们使用HCV2a亚型复制子(JFH-1)侵染Huh7细胞系和Huh7.5.1细胞系,并检测MVP表达水平的变化。结果表明,HCV JFH-1能诱导MVP表达,并呈时间依赖和剂量依赖的方式。为探讨HCV上调MVP表达水平的机理,我们通过PCR的方法获得了MVP基因启动子,将它构建到载体pGL3-basic上。研究发现,HCVNS5A蛋白能够上调MVP启动子的活力,进一步研究表明,转录因子NF-κB和Sp1参与了NS5A对MVP的调控。接着,我们探讨了MVP对HCV的影响。结果表明,MVP能够通过上调Ⅰ型干扰素：nRNA的表达和促进蛋白的分泌,来抑制HCV的复制。进一步研究发现,过表达MVP能够增加IRF7/NF-κB的表达,并且促进IRF7/NF-κB入核,但对IRF3没有什么影响。将MVP干扰后,能够抑制水疱性口炎病毒(VSV)诱导的IRF7/NF-κB的表达和入核,以及Ⅰ型干扰素mRNA的表达和蛋白的分泌。进一步研究发现,水疱性口炎病毒(VSV)、A型流感病毒(IAV)、肠道病毒71(EV71)能诱导MVP的表达,而MVP反过来对这三种病毒都有抑制作用。小结：MVP是一种病毒诱导的宿主因子,它的表达能够上调Ⅰ型干扰素,进而对病毒产生抑制。虽然对MVP在病毒复制和抗病毒反应中的作用的许多细节还不是十分清楚,但是以上研究为揭示MVP的一个新功能(即细胞抵抗病毒作用)提供了新的证据。同时为HCV的治疗奠定理论基础。
[Abstract]:Hepatitis C virus (HCV) infection has become a major public health problem. More than 170 million people worldwide are infected with HCV, of which more than 30 million are infected in China. Major fornix protein (MVP) is a major component of fornix protein, which plays a key role in cell resistance, nuclear and cytoplasmic transport, and cell signaling pathway. So far, the relationship between HCV infection and MVP is unclear. To investigate the relationship between HCV infection and MVP, we detected the difference of MVP mRNA and protein expression between HCV patients and healthy subjects. The results showed that the expression of MVP mRNA in PBMC cells of HCV patients was significantly higher than that in healthy subjects. Similarly, the expression of MVP protein in serum and liver tissues was significantly increased in HCV patients. Because Huh7 cell line and Huh7.5.1 cell line are an effective cell culture model of HCV infection, we used HCV2a subtype replicon (JFH-1) to infect Huh7 cell line and Huh7.5.1 cell line, and to detect the change of MVP expression level. The results showed that HCV JFH-1 could induce MVP expression in a time and dose dependent manner. In order to investigate the mechanism of HCV up-regulation of MVP expression, we obtained the promoter of MVP gene by PCR and constructed it into pGL3-basic vector. It was found that HCVNS5A protein could up-regulate the activity of MVP promoter. Further studies showed that NF- 魏 B and Sp1 were involved in the regulation of MVP by NS5A. Then, we studied the effect of MVP on HCV. The results showed that MVP could inhibit HCV replication by up-regulating the expression of type I interferon: nRNA and promoting protein secretion. Further studies showed that overexpression of MVP increased the expression of IRF7 / NF- 魏 B and enhanced the entry of IRF7 / NF- 魏 B into the nucleus, but had little effect on IRF3. The interference of MVP inhibited the expression and nucleation of IRF7 / NF- 魏 B induced by vesicular stomatitis virus (VSV), as well as the expression of interferon type I mRNA and the secretion of protein. Further studies showed that vesicular stomatitis virus (VSV) influenza A virus (IAV) and enterovirus 71 (EV71) could induce the expression of MVP and MVP could inhibit the expression of MVP. Conclusion: MVP is a virus-induced host factor, which can up-regulate the expression of interferon type I and thus inhibit the virus. Although many details of the role of MVP in viral replication and antiviral response are not well understood, the above studies provide new evidence to reveal a new function of MVP (cell resistance to virus). At the same time, lay a theoretical foundation for the treatment of HCV.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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