模式识别受体介导的小鼠附睾天然免疫反应

发布时间：2018-05-17 08:26

本文选题：附睾 + 模式识别受体　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景与目的：多种病原体(包括细菌、病毒、寄生虫)感染可诱导附睾炎,干扰附睾功能,是损害男性生育能力的重要病因。其中尿路致病性大肠杆菌(Uropathogenic E. coli, UPEC)、腮腺炎病毒(Mumps Virus, MuV)、疱疹病毒(Herpes simplex virus 2, HSV-2)是诱导附睾炎的主要病原体,然而这些病原体诱导附睾炎的机制尚不清楚,本论文以小鼠为模型,旨在研究主要病原体诱导的附睾天然免疫反应的机制。材料与方法：利用TLR2-/-、TLR4-/-、TLR5-/-小鼠模型,结合RNA干扰技术研究相关基因的功能。体外分离与培养原代附睾上皮细胞(epididymal epithelial cells, EECs),用UPEC、MuV与HSV-2感染细胞。利用Real-time RT-PCR实时定量分析基因的mRNA表达水平,Western blot检测基因产物的蛋白水平,免疫组化染色定位蛋白的细胞分布,ELISA检测细胞因子的分泌。附睾原位注射病原体模拟体内感染,分析附睾抵御病原体的天然免疫反应。结果：EEC表达多个模式识别受体,其中TLR4、TLR5可以被UPEC激活并介导EEC的天然免疫反应。TLR2和RIG-I可以被MuV激活,DNA感受器DAI、 p204、cGAS可以被HSV-2诱导上调表达并激活,介导EEC的天然抗病毒反应。UPEC和MuV都可以显著诱导促炎性细胞因子、趋化因子和Ⅰ型干扰素(IFN-α与IFN-p)的表达上调,而HSV-2仅有效上调IFN-α与IFN-β 的表达。在TLR4-/-、TLR5-/-EEC中,附睾抵御UPEC的天然免疫反应都明显低于野生(WT)细胞,说明TLR4与TLR5协同介导UPEC诱导的反应。将TLR2敲除或siRNA沉默RIG-I后,EEC抗MuV病毒的反应也都明显降低,说明TLR2及RIG-I协同介导MuV诱导的天然抗病毒反应。利用siRNA敲低DNA感受器p204、DAI、cGAS时,细胞抗HSV-2病毒能力明显减弱,说明DNA感受器协同介导HSV-2诱导EEC的天然病毒反应。结论：本论文揭示了UPEC,MuV与HSV-2诱导EEC天然免疫反应的模式识别受体及其信号通路,说明附睾组织特异细胞可以调节炎症反应,具有抵抗病原体的天然防御能力。
[Abstract]:Background & objective: infection of many pathogens (including bacteria, viruses and parasites) can induce epididymitis and interfere with epididymal function. Among them, Uropathogenic Escherichia coli, Uropathogenic Escherichia coli, mumps virus Mumps virus, Mumps virus, herpesvirus Herpes simplex virus 2, HSV-2) are the main pathogens to induce epididymitis. However, the mechanism of these pathogens in inducing epididymitis is still unclear. The aim of this study was to study the mechanism of epididymal innate immune response induced by major pathogens. Materials and methods: the function of related genes was studied by using TLR2-P -TLR4-P -TLR5-r-m- mouse model and RNA interference technique. Primary epididymal epithelial cells (EECs) were isolated and cultured in vitro. The cells were infected with UPEC-MuV and HSV-2. The mRNA expression level of the gene was quantitatively analyzed by Real-time RT-PCR and the protein level of the gene product was detected by Western blot. The cell distribution of the protein was determined by immunohistochemical staining and the cytokine secretion was detected by Elisa. In situ injection of pathogens into epididymis simulates in vivo infection and analyzes the innate immune response of epididymis against pathogens. Results TLR4TLR5 could be activated by UPEC and mediated the innate immune response of EEC. TLR2 and RIG-I could be activated by MuV, and p204cGAS could be up-regulated and activated by HSV-2. The natural antiviral response mediated by EEC. UPEC and MuV could significantly induce the expression of pro-inflammatory cytokines, chemokines and IFN- 伪 and IFN-p-), while HSV-2 could only effectively upregulate the expression of IFN- 伪 and IFN- 尾. In TLR4-r-TLR5-EEC, the innate immune response of epididymis to UPEC was significantly lower than that of wild WT-cells, indicating that TLR4 and TLR5 co-mediated the UPEC induced response. After TLR2 knockout or siRNA silencing RIG-I, the antiviral response of EEC was also significantly decreased, indicating that TLR2 and RIG-I co-mediated the MuV induced natural antiviral response. When siRNA knocked down the DNA receptor p204DIAI CGAS, the ability of the cells to resist HSV-2 virus was obviously weakened, indicating that the DNA receptor co-mediated the natural viral response of EEC induced by HSV-2. Conclusion: this study revealed the pattern recognition receptor and its signal pathway of EEC innate immune response induced by HSV-2 and muv, which indicated that specific cells of epididymis could regulate inflammatory response and possess natural defense ability against pathogens.
【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】：R697.22

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