腮腺炎病毒诱导小鼠睾丸天然免疫反应的机制

发布时间：2017-12-31 13:10

本文关键词：腮腺炎病毒诱导小鼠睾丸天然免疫反应的机制　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：背景与目的：腮腺炎病毒(mumps virus, MuV)感染常引发睾丸炎,导致男性不育。虽然疫苗的使用已有效地控制了腮腺炎病毒的大范围流行,但近来临床上腮腺炎病毒感染并发的睾丸炎有复发趋势。认识MuV诱导睾丸天然免疫反应的机制,可为疾病防治提供线索。本文利用小鼠为模型,研究MuV诱导睾丸天然免疫反应的机制及其在睾丸细胞的复制与调控。材料与方法：以C57BL/6或Toll样受体(Toll-like receptor, TLR)基因敲除小鼠(TLR2-/-, TLR3-/-和TLR4-/-)为模型,分离原代睾丸细胞。采用结晶紫染色感染的Vero细胞,测定病毒滴度。实时定量RT-PCR检测基因表达水平,ELISA测定细胞因子的浓度水平,以及Western blot,免疫组织化学和免疫荧光的方法检测蛋白的表达水平与细胞分布。并结合RNA干扰技术研究相关基因的功能。结果：MuV可通过TLR2和RIG-I信号通路诱导Sertoli及Leydig细胞产生天然免疫反应,产生TNF-α、IL-6、MCP-1、CXCL10及Ⅰ型干扰素(IFN-α和IFN-p)等细胞因子,而不能诱导生精细胞产生这些细胞因子。MuV感染后,Sertoli细胞产生的炎症因子及趋化因子水平较Leydig细胞高,但IFN-a与IFN-β水平要低于Leydig细胞。体内实验证实,睾丸原位感染MuV可诱发睾丸局部天然免疫反应,并抑制Leydig细胞合成睾酮。MuV能够感染多种睾丸细胞,包括Sertoli、Leydig、巨噬细胞和生精细胞,但MuV在不同细胞中的复制效率存在差异,这与细胞特异的抗病毒防御机制有关。MuV在Sertoli细胞中能快速复制并持续存在,在Leydig细胞和巨噬细胞中也有显著性的复制,但在生精细胞中则检测不到病毒的复制。Sertoli, Leydig,和巨噬细胞主要依靠产生Ⅰ型干扰素和抗病毒蛋白来抵御病毒感染,生精细胞则是依赖自身高水平的自噬来限制病毒的复制。结论：MuV能够诱导睾丸Sertoli及Leydig细胞产生天然免疫反应,且这一过程由TLR2和RIG-I信号通路介导。MuV可以感染睾丸细胞并进行复制,受不同细胞的抗病毒机制调节。以上结果为认识MuV诱导睾丸细胞的天然免疫反应及睾丸的抗病毒防御机制提供了新思路。
[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- 伪, IL-6, MCP-1, CXCL10 and IFN- 伪 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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

【相似文献】