Sohlh2/c-kit信号通路在高温抑制精原干细胞分化中作用及作用机制的研究

High temperature is one of factors which induced male infertility. Our group found that high temperature in environment inhibited sohlh2 and c-kit gene expression, which regulated SSCs differentiation. We speculated that high temperature might block sohlh2/c-kit pathway and inhibit SSCs differentiation, but the mechanism was rarely reported.Therefore, we intend to adopt the high-temperature to treat the in vitro cultured SSCs and in vivo SSCs to verify inhibition of high temperature for SSCs differentiation by flow cytometry , real-time PCR and western blot in this study. We investigate the effect of c-kit and sohlh2 genes over expression on the recovery of SSCs differentiation, which are blocked by high-temperature, by Plenti-c-kit infection and SSCs transplantation technologies. We analyze the regulatory genes of sohlh2 which could inhibit sohlh2/c-kit signaling pathway when the expression is downregulated. In addition, we investigate the relationship between the heat shock transcription factor1（HSF1）and sohlh2/c-kit signaling pathway.With these studies, the mechanism of infertility caused by high temperature will be revealed, and the theoretical and experimental basis for the treatment of infertility in clinics can be provided.

环境高温所致的精子损伤及发生障碍是造成男性不育的因素之一。本课题组研究发现高温抑制精原干细胞（SSCs）分化，下调SSCs分化通路sohlh2/c-kit关键基因sohlh2和c-kit的表达，故假设环境高温通过阻断sohlh2/c-kit通路抑制了SSCs分化，但相关研究鲜见报道。因此本研究拟采用在体SSCs和体外培养SSCs，通过流式细胞仪、real-time PCR以及western blot等技术手段研究sohlh2/c-kit通路在高温抑制SSCs分化中的作用；应用慢病毒过表达和RNA干扰载体及SSCs移植技术，进一步明确该通路在高温抑制SSCs分化中的特异性；采用基因芯片技术筛选、分析以及明确高温下调sohlh2基因表达的关键基因及分子机制；研究热休克转录因子HSF1与该通路之间的关系，从而探明高温抑制SSCs分化的分子调控机制，为男性不育的基因治疗以及新药研发提供科学依据。

环境高温所致的精子损伤及发生障碍是造成男性不育的因素之一。本研究采用幼龄和成年小鼠高温热应激处理模型，发现高温抑制在体精原干细胞（SSCs）分化，下调精原干细胞分化通路sohlh2/c-kit中sohlh2和c-kit 基因表达，精母细胞数量减少，睾丸初级精母细胞表面标记蛋白SYCP3基因与蛋白表达水平降低。采用Bmp4、维甲酸、bFGF 等因子可激活体外培养精原干细胞 Sohlh2/c-kit干细胞分化调控通路，成功地建立了精原干细胞体外分化培养体系；采用体外分化精原干细胞热应激处理模型，发现高温能够下调c-kit基因，抑制精原干细胞分化。采用慢病毒载体过表达sohlh2基因后促进了精原干细胞分化，且能逆转高温对精原干细胞分化的抑制作用。采用转录组测序技术，检测到高温抑制精原干细胞增殖相关差异表达基因200个，并通过KEGG富集分析与荧光定量PCR验证得到与精原干细胞增殖抑制和周期阻滞密切相关的信号通路Jak-STAT 信号通路，采用流式细胞仪技术进一步发现热应激处理造成了精原干细胞S期周期阻滞。调控高温处理后热休克蛋白表达的热休克转录因子 HSF1 在高温热应激处理后表达无差异，表明与干细胞增殖没有关联。本研究初步阐明了高温抑制精原干细胞分化的调控机制，为高温不育症的诊治提供参考。