TLR3在小鼠生精细胞中的表达及功能

发布时间：2019-01-04 07:51

【摘要】：背景与目的：睾丸是典型的免疫豁免组织,却能有效的清除感染的病原体,睾丸细胞的天然免疫防御功能长期受到研究者的关注,其调节机理有待进一步揭示。Toll样受体(TLR)可以启动睾丸体细胞的天然免疫反应,然而在生精细胞中的表达和功能尚未做深入研究。本论文旨在系统研究TLR3在生精细胞中的表达和功能。材料与方法：分离止常C57BL/6小鼠各期生精细胞,使用其标记基因,包括精原细胞的标记基因Kit,精母细胞的标记基因联会复合体蛋白(synaptonemal complex protein2,Scp2),精子细胞的标记基因鱼精蛋白2(protamin2, Prm2);通过real-time RT-PCR,免疫组化及Western blotting技术分别在mRNA及蛋白水平上检测TLR3在生精细胞中的表达；利用poly(I:C)激活生精细胞中的TLR3研究其在生精细胞中的功能及信号通路。吖啶橙／溴化乙锭(AO/EB)混合染色,TUNEL染色及流式细胞仪分析生精细胞的凋亡。结果：发现小鼠生精细胞组成性的表达TLR3,主要定位于精原细胞和精母细胞,poly(I:C)可诱导精原和精母细胞产生多种炎症因子(包括IL-1p,IL-6和TNF-a),1型干扰素(IFNa, IFNβ)及抗病毒蛋白(OAS1, PKR, MX1),这种作用依赖于NF-κB和IRF-3的激活。另外TLR3的激活还可以诱导生精细胞的凋亡,这种作用可能是通过上调TNF-α实现的。体外实验证明TNF-α表现出双重功能：低浓度的TNF-α可以抑制生精细胞的凋亡,而高浓度的TNF-α可以促进生精细胞的凋亡。结论：TLR3表达于精原与精母细胞,生精细胞中TLR3的激活可以行使两方面的功能：一方面启动生精细胞抗病毒的天然免疫反应,另一方面可以诱导生精细胞的凋亡。为病毒感染干扰精子发生的机理提供了新的线索。
[Abstract]:Background & objective: testis are typical immune exempted tissues, but they can effectively remove the infected pathogens. The innate immune defense function of testicular cells has been concerned by researchers for a long time. Toll like receptor (TLR) can initiate the innate immune response of somatic cells in testis, but its expression and function in spermatogenic cells have not been further studied. The purpose of this thesis is to study the expression and function of TLR3 in spermatogenic cells. Materials and methods: spermatogenic cells of normal C57BL/6 mice were isolated and labeled with their marker genes, including the marker gene of spermatogonial Kit, (synaptonemal complex protein2,Scp2. Sperm cell labeled gene protamine 2 (protamin2, Prm2); The expression of TLR3 in spermatogenic cells was detected by real-time RT-PCR, immunohistochemistry and Western blotting technique at the level of mRNA and protein respectively. Poly (I: C) was used to activate TLR3 in spermatogenic cells to study its function and signal pathway in spermatogenic cells. Acridine orange / ethidium bromide (AO/EB) mixed staining, TUNEL staining and flow cytometry were used to analyze the apoptosis of spermatogenic cells. Results: the constitutive expression of TLR3, in mouse spermatogenic cells was mainly localized in spermatogonia and spermatocyte, poly (I: C), which could induce the production of many inflammatory factors (including IL-1p,IL-6 and TNF-a) in spermatogonial and spermatogenic cells. Type 1 interferon (IFNa, IFN 尾) and antiviral protein (OAS1, PKR, MX1), which depends on the activation of NF- 魏 B and IRF-3. In addition, the activation of TLR3 can also induce the apoptosis of spermatogenic cells, which may be achieved by upregulating TNF- 伪. In vitro experiments showed that TNF- 伪 had dual functions: low concentration of TNF- 伪 could inhibit the apoptosis of spermatogenic cells, while high concentration of TNF- 伪 could promote the apoptosis of spermatogenic cells. Conclusion: TLR3 is expressed in spermatogonial and spermatogenic cells. The activation of TLR3 in spermatogenic cells can perform two functions: on the one hand, it can activate the innate immune response of spermatogenic cells against virus, on the other hand, it can induce the apoptosis of spermatogenic cells. It provides a new clue for virus infection to interfere the mechanism of spermatogenesis.
【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】：2012
【分类号】：R321

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