【作者】 张玥；

【导师】 韩代书；

【作者基本信息】 北京协和医学院 ， 细胞生物学， 2013， 博士

【摘要】 背景与目的：睾丸是一个免疫豁免组织,这种免疫豁免环境对精子发生具有重要的保护作用。TAM受体三基因敲除(TAM-/-)小鼠雄性不育,然而对TAM受体调节雄性小鼠生育力的作用机理尚未做深入研究。本论文旨在分析TAM受体在调节睾丸免疫豁免稳态中的作用。材料与方法：利用TAM受体敲除小鼠为模型。利用组织学方法分析睾丸结构与精子发生；免疫组织化学和流式细胞术分析睾丸间质免疫细胞的浸润；电镜和生物素示踪方法分析血睾屏障的结构及通透性；Western blot和免疫组化染色研究蛋白的表达及定位。分离纯化小鼠Sertoli细胞以及生精细胞,利用实时定量PCR分析基因mRNA的水平,ELISA检测炎症因子的水平。结果：TAM-/-小鼠的精子发生在性成熟以后随着鼠龄的增加逐步受损。TAM-/-小鼠睾丸间质中的免疫细胞数量显著高于野生型小鼠,在20和30周龄的TAM-/-小鼠的曲细精管管腔中也观察到巨噬细胞的浸润；血睾屏障结构被破坏,对生物素的通透性增加；随着鼠龄的增长产生抗生精细胞的自身抗体。睾丸组织中炎症因子TNF-α、 IL-6、MCP-1的表达升高；表达上调的炎症因子主要定位于Sertoli细胞。体外实验表明,凋亡的生精细胞能够诱导Sertoli细胞产生炎症反应；这种炎症反应在TAM-/-Sertoli细胞显著高于野生型Sertoli细胞。结论：TAM受体通过调节Sertoli细胞的功能在维持睾丸免疫稳态中起到重要的作用,敲除TAM可导致自发性慢性睾丸炎,从而影响雄性小鼠的生育力。更多还原

【Abstract】 Background and objectives:The testis is a remarkable immune-privileged site that is necessary to protect the germ cells from systemic immune attack, which is critical for normal spermatogenesis. Tyro3, Axl and Mer (TAM) receptor tyrosine kinases triple knockout (TAM-/-) mice are male sterile. However, the mechanism that TAM receptors regulate male fertility remains unclear. In this study, we examine the role of TAM receptors in maintaining testicular immune homeostasis.Materials and methods:TAM-/-mice were used in this study. Testicular structure and spermatogenesis were examined by histology after hematoxylin-eosin staining. The infiltrations of immune cells into the testis were analyzed using flow cytometry and immunohistochemistry. Electron microscopy and biotin tracer assay were used to examine the structure and permeability of blood-testis barrier (BTB). Sertoli cells and germ cells were isolated. Gene expression was examined using qRT-PCR, Western blot, ELISA and immunohistochemistry.Results:A chronic orchitis is developed in adult TAM-/-mice. As mice aging after the onset of sexual maturity, germ cells were progressively degenerated. Coincidently, macrophage infiltration was observed in the testis of TAM-/-mice ages20and30weeks. Moreover, the integrity of BTB was impaired, and the autoantibodies against germ cell antigens were generated as TAM-/-mice aged. The upregulation of major pro-inflammatory cytokines, including TNF-a, IL-6, and monocyte chemotactic protein1, was predominantly observed in Sertoli cells (SCs). In vitro assays showed that SCs lacking TAM receptors secrete relatively high levels of pro-inflammatory cytokines compared with wild-type controls in response to apoptotic germ cells.Conclusions:These results suggest that TAM receptors play an important role in the maintenance of the immune homeostasis in the testis by regulating SC functions. TAM-/-mice developed chronic orchitis, thus impairing male fertility.更多还原