【作者】 张勇；

【导师】 杨为民；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 目的：建立睾丸支持细胞体外缺氧模型，研究缺氧对于雄性生殖系统中支持细胞的活性和功能的影响，在此基础之上进一步探讨产生活性和功能影响的可能机制，并寻找能够减缓缺氧损伤的保护剂。方法：在睾丸支持细胞体外缺氧模型的基础之上，利用CoCl2作为体外缺氧模型的模拟剂。模拟支持细胞的缺氧环境，采用MTT和流式细胞仪检测缺氧状态下支持细胞的活性和凋亡率。并利用CoCl2模拟不同浓度下的支持细胞活性变化，检测细胞活性是否存在剂量依赖性和时间依赖性。利用实时定量荧光PCR检测支持细胞凋亡情况，Western blot检测凋亡效应分子caspase-3的变化。Real-time PCR检测支持细胞的分泌功能。结果：成功建立体外缺氧模型，支持细胞形态改变，细胞发生皱缩，体积缩小。发现CoCl2诱导缺氧能降低支持细胞活性存在浓度依赖性，但是不具备时间依赖性。同样对于支持细胞凋亡也存在浓度依赖性，而且也具备时间依赖性。CoCl2诱导缺氧能够通过氧化应激引起依赖线粒体的凋亡途径激活导致支持细胞凋亡。并且能够激活促丝裂原活化蛋白激酶途径：缺氧能够诱导原代支持细胞凋亡，通过氧化应激形式，在促丝裂原蛋白激酶途径的转导作用下激活依赖线粒体的内源性凋亡途径。缺氧还能够引起支持细胞功能发生改变，可能继而影响生精功能。结论：成功建立支持细胞体外缺氧模型，发现支持细胞在缺氧诱导剂作用下存在细胞活性和凋亡率的剂量依赖性改变。而MAPK信号通路参与了凋亡信号的转导。凋亡由氧化应激激活的内源性凋亡途径完成。更多还原

【Abstract】 Objective: To construct testicular Sertoli cell hypoxic model in vitro. We plan toinvestigate the function and apoptosis of Sertoli cells in the male reproductive system inhypoxia state and speculate about the possible effect of hypoxia on the reproductive system.And we want to serch protective agent of hypoxia.Methods: On the basis of hypoxia of Sertoli cells induced by CoCl2in vitro, we detect cellactivity and spoptosis of Sertoli cells under hypoxia by MTT assay and flow cytometryinstrument. And under different concentrations of CoCl2simulation Sertoli cells activitychanges. We detect apoptosis of Sertoli cells by real-time PCR, and chang of apoptoticeffect molecules caspase-3by Western blot. And we detect secrete function of Sertoli cellsunder hypoxia.Results: We construct cell hypoxia model in vitro. Morphological changes, cell shrinkagehappened under hypoxia. Activity of Sertoli cells under hypoxia reduced bydose-dependent, but not time-dependent. For Sertoli cells apoptosis is concentrationdependent, and have time denpendece. Hypoxia induced by CoCl2can result Sertoli cellsapoptosis by relying on mitochondrial apoptosis pathway. And it was able to activate andpromote MAPK signal pathway. Hypxoa can induce apoptosis of primary Sertoli cells byoxidative stress form. Hypoxia also can induce a function change of Sertoli cells. After thatit can affect sperm production.Conclusion: We can construct cell hypoxia model of Sertoli cells in vitro successfully andwe found that Sertoli cells have dose dependence of cell activity and apoptosis rate change.And MAPK signal pathway involved in apoptosis signal transduction. Apoptosis ofactivated by oxidative stress intrinsic apoptotic pathways.更多还原