模式识别受体介导的小鼠睾丸和附睾的天然抗病毒反应

发布时间：2018-01-18 07:19

本文关键词：模式识别受体介导的小鼠睾丸和附睾的天然抗病毒反应　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：背景与目的：病毒感染引起的睾丸炎和附睾炎,是破坏男性生育能力的重要因素。目前这两个生殖器官的天然抗病毒免疫机制尚不清楚,本论文主要研究模式识别受体介导的睾丸与附睾天然抗病毒机制,为预防和治疗相关疾病提供新线索。材料与方法：以小鼠为模型,用poly(I:C)和HSV60模拟病毒感染,利用实时定量RT-PCR检测基因的mRNA水平,Western blot测定基因的蛋白水平,免疫组化染色定位蛋白的分布,ELISA检测细胞因子和睾酮的分泌。利用TLR3基因敲除(TLR3-/-)小鼠和RNA干扰技术确证基因的功能。结果：睾丸Leydig细胞组成性表达病毒核酸受体MDA5、RIG-Ip204,其中MDA5也在圆形和长形精子中表达。MDA5和RIG-I可以被poly(I:C)激活,p204可以被HSV60激活,诱导睾丸抗病毒反应。附睾上皮细胞组成性表达多个病毒核酸受体,包括TLR3、RIG-I和DAI。p204与cGAS可以被HSV60诱导表达。TLR3和RIG-I可以被poly(I:C)激活,而DAI、p204 与 cGA S可以被HSV60激活,启动附睾天然抗病毒反应。在睾丸及附睾细胞中,poly(I:C)和 HSV60都能诱导Ⅰ型干扰素和抗病毒蛋白ISG15、OAS1和MX1的表达。poly(I:C)可以显著上调炎症因子TNF-α和MCP-1的表达,但HSV60并不影响TNF-a和MCP-1的表达。在TLR3-/-细胞中或用siRNA敲低RIG-I、DAI、p204或cGAS时,抗病毒反应明显减弱,说明这几个受体协同介导睾丸及附睾的天然抗病毒反应。结论：在睾丸中,MDA5/RIG-I启动抗RNA病毒的反应,而p204介导抗DNA病毒的天然免疫反应；在附睾中,TLR3与RIG-I介导抗RNA病毒的反应,DAI、p204和cGAS参与抗DNA病毒的反应。以上结果说明睾丸与附睾组织特异细胞具有完善的天然抗病毒能力。
[Abstract]:Background & objective: orchitis and epididymitis caused by virus infection are important factors to destroy male fertility. At present, the natural antiviral immune mechanism of these two reproductive organs is not clear. This paper mainly studies the natural antiviral mechanism of testis and epididymis mediated by pattern recognition receptor, which provides a new clue for the prevention and treatment of related diseases. Materials and methods: mouse model. The viral infection was simulated by polymorphic I: C) and HSV60. The mRNA level of the gene was detected by real-time quantitative RT-PCR and the protein level of the gene was determined by Western blot. Immunohistochemical staining was used to locate the distribution of proteins. ELISA was used to detect the secretion of cytokines and testosterone. TLR3 gene knockout was used to remove TLR3 / -). Mouse and RNA interference techniques confirmed the function of the gene. Results: testicular Leydig cells expressed viral nucleic acid receptor MDA5 constitutively. RIG-Ip204, in which MDA5 is also expressed in round and long spermatozoa. MDA5 and RIG-I can be activated by polypeptide I: C) and p204 can be activated by HSV60. Induces testicular antiviral response. Epididymal epithelial cells constitutively express multiple viral nucleic acid receptors, including TLR3. RIG-I and DAI.p204 and cGAS can be induced by HSV60. TLR3 and RIG-I can be activated by polycyclic C) and DAI. P204 and cGA S can be activated by HSV60, initiating the natural antiviral reaction of epididymis, in testicular and epididymal cells. Polymorphic I: C) and HSV60 can induce interferon type I and antiviral protein ISG15. The expression of OAS1 and MX1 could significantly up-regulate the expression of TNF- 伪 and MCP-1. However, HSV60 did not affect the expression of TNF-a and MCP-1. In TLR3-r-cells or when siRNA was used to knock down RIG-Idae P204 or cGAS. The antiviral response was significantly weakened, indicating that these receptors co-mediated the natural antiviral response of testis and epididymis. Conclusion: MDA5 / RIG-I initiates the anti-viral response in testis. P204 mediated innate immune response against DNA virus. In epididymis, TLR3 and RIG-I mediate the response to RNA virus. P204 and cGAS are involved in the response to DNA virus. These results suggest that testicular and epididymal tissue specific cells have perfect natural antiviral ability.
【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】：2015
【分类号】：R698.2

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