發(fā)布時(shí)間：2017-12-31 13:10

  本文關(guān)鍵詞：腮腺炎病毒誘導(dǎo)小鼠睪丸天然免疫反應(yīng)的機(jī)制　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腮腺炎病毒 睪丸 模式識(shí)別受體 天然免疫反應(yīng) 自噬

【摘要】：背景與目的：腮腺炎病毒(mumps virus, MuV)感染常引發(fā)睪丸炎,導(dǎo)致男性不育。雖然疫苗的使用已有效地控制了腮腺炎病毒的大范圍流行,但近來臨床上腮腺炎病毒感染并發(fā)的睪丸炎有復(fù)發(fā)趨勢(shì)。認(rèn)識(shí)MuV誘導(dǎo)睪丸天然免疫反應(yīng)的機(jī)制,可為疾病防治提供線索。本文利用小鼠為模型,研究MuV誘導(dǎo)睪丸天然免疫反應(yīng)的機(jī)制及其在睪丸細(xì)胞的復(fù)制與調(diào)控。材料與方法：以C57BL/6或Toll樣受體(Toll-like receptor, TLR)基因敲除小鼠(TLR2-/-, TLR3-/-和TLR4-/-)為模型,分離原代睪丸細(xì)胞。采用結(jié)晶紫染色感染的Vero細(xì)胞,測(cè)定病毒滴度。實(shí)時(shí)定量RT-PCR檢測(cè)基因表達(dá)水平,ELISA測(cè)定細(xì)胞因子的濃度水平,以及Western blot,免疫組織化學(xué)和免疫熒光的方法檢測(cè)蛋白的表達(dá)水平與細(xì)胞分布。并結(jié)合RNA干擾技術(shù)研究相關(guān)基因的功能。結(jié)果：MuV可通過TLR2和RIG-I信號(hào)通路誘導(dǎo)Sertoli及Leydig細(xì)胞產(chǎn)生天然免疫反應(yīng),產(chǎn)生TNF-α、IL-6、MCP-1、CXCL10及Ⅰ型干擾素(IFN-α和IFN-p)等細(xì)胞因子,而不能誘導(dǎo)生精細(xì)胞產(chǎn)生這些細(xì)胞因子。MuV感染后,Sertoli細(xì)胞產(chǎn)生的炎癥因子及趨化因子水平較Leydig細(xì)胞高,但I(xiàn)FN-a與IFN-β水平要低于Leydig細(xì)胞。體內(nèi)實(shí)驗(yàn)證實(shí),睪丸原位感染MuV可誘發(fā)睪丸局部天然免疫反應(yīng),并抑制Leydig細(xì)胞合成睪酮。MuV能夠感染多種睪丸細(xì)胞,包括Sertoli、Leydig、巨噬細(xì)胞和生精細(xì)胞,但MuV在不同細(xì)胞中的復(fù)制效率存在差異,這與細(xì)胞特異的抗病毒防御機(jī)制有關(guān)。MuV在Sertoli細(xì)胞中能快速復(fù)制并持續(xù)存在,在Leydig細(xì)胞和巨噬細(xì)胞中也有顯著性的復(fù)制,但在生精細(xì)胞中則檢測(cè)不到病毒的復(fù)制。Sertoli, Leydig,和巨噬細(xì)胞主要依靠產(chǎn)生Ⅰ型干擾素和抗病毒蛋白來抵御病毒感染,生精細(xì)胞則是依賴自身高水平的自噬來限制病毒的復(fù)制。結(jié)論：MuV能夠誘導(dǎo)睪丸Sertoli及Leydig細(xì)胞產(chǎn)生天然免疫反應(yīng),且這一過程由TLR2和RIG-I信號(hào)通路介導(dǎo)。MuV可以感染睪丸細(xì)胞并進(jìn)行復(fù)制,受不同細(xì)胞的抗病毒機(jī)制調(diào)節(jié)。以上結(jié)果為認(rèn)識(shí)MuV誘導(dǎo)睪丸細(xì)胞的天然免疫反應(yīng)及睪丸的抗病毒防御機(jī)制提供了新思路。
[Abstract]:Background & objective: mumps virus (Muv) infection often causes orchitis. Causes male infertility. Although the use of the vaccine has been effective in controlling the widespread prevalence of mumps virus. But recently, the mumps virus infection complicated with orchitis has a tendency to recur. Understanding the mechanism of MuV induced testicular innate immune response can provide clues for the prevention and treatment of the disease. To study the mechanism of testicular innate immune response induced by MuV and its replication and regulation in testicular cells. Materials and methods: C57BL / 6 or Toll like receptor (C57BL / 6). Toll-like receptor. Primary testicular cells were isolated from TLR2-r-, TLR3-r- and TLR4-r- by using crystal violet staining of Vero cells. The viral titer was measured. The gene expression level was detected by real-time quantitative RT-PCR. The concentration of cytokines was measured by Elisa, and the Western blot was measured. Immunohistochemical and immunofluorescence methods were used to detect the expression level and cell distribution of proteins. The function of related genes was studied by RNA interference technique. Results:. MuV can induce the innate immune response of Sertoli and Leydig cells through TLR2 and RIG-I signaling pathway. Cytokines such as TNF- 偽, IL-6, MCP-1, CXCL10 and IFN- 偽 and IFN-p were produced. However, the levels of inflammatory and chemokines produced by Sertoli cells were higher than those of Leydig cells. However, the levels of IFN-a and IFN- 尾 were lower than those of Leydig cells. In vivo, testicular in situ infection with MuV could induce local innate immune response of testis. Inhibiting the synthesis of testosterone by Leydig cells can infect many testicular cells, including Sertoli Leydigs, macrophages and spermatogenic cells. However, the replication efficiency of MuV in different cells is different, which is related to the cell-specific anti-virus defense mechanism. MuV can replicate rapidly and persist in Sertoli cells. There was also significant replication in Leydig cells and macrophages, but not in spermatogenic cells. Sertoli, Leydig was not detected in spermatogenic cells. Macrophages and macrophages mainly rely on the production of interferon type I and antiviral proteins to resist viral infection. Spermatogenic cells are dependent on high levels of autophagy to limit the replication of the virus. Conclusion: Muv can induce the innate immune response of Sertoli and Leydig cells in testis. And this process is mediated by TLR2 and RIG-I signaling pathway. MuV can infect testicular cells and replicate. The above results provide a new idea for understanding the innate immune response of testicular cells induced by MuV and the antiviral defense mechanism of testis.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

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