基质金属蛋白酶3-MMP3在抗登革病毒感染中的作用及机制的研究

发布时间：2018-01-22 21:06

本文关键词： 登革病毒 MMP3 NFκB　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：登革病毒是当今世界上分布最广的一种虫媒传染病毒，主要由叮咬人的埃及伊蚊和白纹伊蚊传播。据WHO估计，全球每年新增数百万的登革病毒感染者，因登革病毒感染导致死亡的人数约2.5万。目前，登革病毒感染的机制仍不明确，由于各血清型之间缺乏有效的交叉保护，又存在抗体依赖的增强作用等，至今仍无安全有效的疫苗可用。因此，研究宿主抗登革病毒的分子机制，，对于了解登革热的发病过程具有重要意义。我们实验室从JAK-STAT信号通路入手，来研究宿主细胞抗登革病毒感染的分子机制，着重关注包括基质金属蛋白酶3（MMP3）在内的几个JAK-STAT下游效应分子在抗登革病毒感染中的具体作用。我们研究在登革病毒感染巨噬细胞前后，84个JAK-STAT相关基因的表达变化。结果显示基质金属蛋白酶MMP3表达上调了3.68倍。为了进一步验证PCR芯片的结果，我们又用定量PCR的方法在多种细胞中证明了MMP3在登革病毒感染的细胞中表达上调。抑制MMP3的表达，病毒的感染水平显著上升；而在RAW264.7细胞中过表达MMP3又可以抑制登革病毒的复制，这就揭示MMP3具有抗登革病毒感染的作用。为了了解MMP3抗病毒的分子机制，我们分别检测了MMP3沉默的细胞和对照细胞经登革病毒感染后，代表性抗病毒相关细胞因子的表达。结果发现，MMP3沉默的细胞中，抗病毒相关细胞因子和趋化因子的表达水平显著降低。免疫荧光发现MMP3在登革病毒感染的细胞中会发生从细胞质到细胞核的迁移；而且双荧光素酶报告基因检测结果显示，在病毒感染的细胞中，沉默掉MMP3，NFκB的转录活性下调，过表达MMP3又可以很强地激活NFκB。鉴于以上这些结果，我们推测MMP3很有可能是通过进入细胞核来影响NFκB的转录活性从而达到抗病毒作用。在后续实验中通过采用荧光定位和免疫共沉淀的方法得到了MMP3与NFκB在登革病毒感染的细胞中存在相互作用的结果。 MMP3由信号域，前肽域，催化域，铰链区和羧基末端域等5个结构域组成，为了检测与NFκB相互作用的结构域，我们构建了含MMP3各个结构域的质粒，并分别与NFκB的两个亚基（P50; P65）共转染293T细胞进行免疫共沉淀实验。结果显示MMP3主要依赖于其羧基末端域与NFκB相互作用。进一步的报告基因检测实验结果显示：MMP3的羧基末端域可以更强地激活NFκB。本研究首次揭示了MMP3进入细胞核发挥抗病毒效应的全新功能，为MMP家族参与免疫调控提供新的证据，而且MMP3分子的表达水平有可能成为登革热疾病进程的诊断靶标，MMP3相关激动剂也将有可能应用到抗病毒感染的应用当中。
[Abstract]:Dengue virus is one of the most widely distributed insect-borne viruses in the world. It is mainly transmitted by Aedes aegypti and Aedes albopictus, which bite people. According to WHO estimates, millions of new dengue virus infections occur every year in the world. At present, the mechanism of dengue virus infection is still unclear, due to the lack of effective cross-protection among the serotypes, and the enhancement of antibody dependence. So far, there is no safe and effective vaccine available. Therefore, it is important to study the molecular mechanism of host versus dengue virus in understanding the pathogenesis of dengue fever. Our laboratory studies the molecular mechanism of host cells against dengue virus infection from the JAK-STAT signaling pathway. Highlights include matrix metalloproteinase 3 (MMP3). We studied the specific role of several downstream JAK-STAT effectors in anti-dengue virus infection before and after dengue virus infection in macrophages. The expression changes of 84 JAK-STAT related genes. The results showed that the expression of matrix metalloproteinase MMP3 increased by 3.68 times. In order to further verify the results of PCR chip. We also demonstrated that the expression of MMP3 in dengue virus infected cells was upregulated by quantitative PCR, and the expression of MMP3 was inhibited, and the level of viral infection was significantly increased. Overexpression of MMP3 in RAW264.7 cells can inhibit the replication of dengue virus, which indicates that MMP3 has the effect of anti-dengue virus infection. In order to understand the molecular mechanism of MMP3 antivirus, we detected the expression of representative antiviral cytokines in MMP3 silenced cells and control cells after dengue virus infection. In the silenced cells of MMP3. The expression of antiviral related cytokines and chemokines decreased significantly. Immunofluorescence showed that MMP3 migration from cytoplasm to nucleus occurred in dengue virus infected cells. The results of double luciferase reporter gene detection showed that the transcription activity of MMP3NF-kappa B was down-regulated in virus-infected cells. Overexpression of MMP3 can strongly activate NF 魏 B. in view of these results. We speculate that MMP3 may affect the transcription activity of NF 魏 B by entering the nucleus to achieve antiviral effect. In subsequent experiments, MM was obtained by fluorescence localization and immunoprecipitation. P3 and NF 魏 B interact with each other in dengue virus infected cells. MMP3 consists of five domains: signal domain, prepeptide domain, catalytic domain, hinge domain and carboxyl terminal domain. We constructed the plasmids containing various domains of MMP3 and associated with two subunits of NF 魏 B, P50; P65). Cotransfection of 293T cells was carried out by immunoprecipitation assay. The results showed that MMP3 mainly depended on the interaction between its carboxyl terminal domain and NF 魏 B. The carboxyl terminal domain of MMP3 can activate NF 魏 B more strongly. This study revealed for the first time that MMP3 enters the nucleus to play a new role in antiviral effect, providing new evidence for MMP family to participate in immune regulation. Moreover, the expression level of MMP3 may become a diagnostic target for dengue disease progression. MMP3 related agonists may also be used in antiviral infection.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.8

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