卵泡微环境iNOS致颗粒细胞增殖分化障碍的机制研究

Proliferation and differentiation disorder of granulosa cells is the key role for infertility which induced by chronic stress. By the means of glucocorticoid model which is the chronic stress simulation model,we showed that: glucocorticoids inhibited proliferation and differentiation of granulosa cells，and granulosa cells in normal mice responded well to FSH challenge by proliferation and differentiation promoted,however granulosa cells in mice stimulated by glucocorticoids responded poorlier to FSH than that of normal mice. This indicates a defect in ovary microenvirnment that is at the post FSH signal events. The further research showed that glucocorticoids downregulated FSHR expression and upregulated iNOS expression,then inhibition of iNOS blocked downregulation of FSHR induced by glucocorticoids. This imply that iNOS in follicular microenvironment is the critical role in proliferation and differentiation disorder of granulosa cells. Bioinformatics analysis shows that CREB is a transcription factor of FSHR,and inhibition of iNOS upregulated CREB phosphorylation. In our study,we will investigate the impact of chronic stress on proliferation and differentiation of granulosa cells and the the impact on related molecular-iNOS and FSHR expression. Then we will get the pros and cons of evidence of the abnormal process in proliferation and differentiation of granulosa cells. At the end we will explain the mechanism that iNOS induces the abnormal proliferation and differentiation process of granulosa cells. This thoery did not be reported,and it is original. If the theory is confirmed, it will represent a novel strategy for the treatment of infertility.

卵巢颗粒细胞（GC）增殖分化障碍在慢性应激致不孕症事件中发挥关键作用。前期发现，糖皮质激素刺激（慢性应激）致GC增殖分化异常；正常小鼠接受FSH刺激后颗粒细胞增殖分化显著加强，但糖皮质激素刺激后小鼠GC增殖分化对FSH的反应远不及正常小鼠反应，提示FSH分子下游的卵巢微环境存在糖皮质激素的调控位点。进一步发现，糖皮质激素上调卵泡微环境iNOS表达、下调FSHR表达，而iNOS抑制剂能阻断FSHR下调，提示卵泡微环境iNOS可能是调控GC增殖分化的重要因素。生物信息学分析提示CREB是FSHR的转录因子，给予iNOS抑制剂可上调CREB的磷酸化。本研究拟观察应激对GC增殖分化及iNOS、FSHR表达的影响；从正反两方面获得iNOS影响颗粒细胞增殖分化的充分证据，并探讨其机制。卵泡微环境iNOS致颗粒细胞增殖分化障碍的研究未见报道，本课题有源头创新性，将可能为不孕症治疗提供新的干预靶点

慢性应激相关的不孕症发病率逐年增加，严重影响了女性的生活和工作，为社会带来巨大的负担。慢性应激状态下的高糖皮质激素不仅改变了颗粒细胞分泌雌孕激素的功能，还影响了卵泡的发育，造成生殖系统功能改变。对这一现象的探讨和研究将为治疗慢性应激相关的生殖疾病提供新的思路。. 本研究从细胞水平和整体动物水平观察高浓度/剂量糖皮质激素对黄素化颗粒细胞功能和形态以及卵泡发育的影响。高浓度皮质酮刺激体外培养的黄素化颗粒细胞合成和分泌孕激素增加，合成雌激素减少，但雌激素分泌增加。延长细胞培养时间，高浓度皮质酮依然刺激孕激素的合成和分泌，但是雌激素的合成和分泌均减少。高浓度皮质酮诱导颗粒细胞的数量增加，对颗粒细胞的增殖没有显著性影响，但明显减少颗粒细胞的凋亡。上述研究结果分别从颗粒细胞功能和形态学角度阐述了皮质酮的作用，由此可推测糖皮质激素对黄体可能具有增强黄体功能，促进孕激素合成分泌作用；另外尚可减少颗粒细胞凋亡，延长黄体生命周期。若这一理论在整体能被证实，则可有望用于排卵后黄体功能不全的黄体支持治疗。. 体内研究发现高剂量皮质酮持续21天刺激小鼠后，卵巢卵泡发育发生改变，即卵泡总数减少，卵泡募集减少，静息原始卵泡增加，卵泡闭锁减少。从皮质酮对卵泡发育的影响看，皮质酮减少了原始卵泡募集启动进入发育环节，另外，卵泡闭锁减少这一结果和体外细胞皮质酮减少颗粒细胞凋亡结果相一致。PMSG是辅助生殖常用药物，可促进卵泡发育。当PMSG刺激糖皮质激素作用的卵泡时，卵泡池中原始卵泡闭锁消亡减少，更多的原始卵泡进入卵泡募集状态，提示PMSG弱化了皮质酮对卵泡发育的负性作用。. 综合上述结果推测糖皮质激素对排卵前卵泡发育具有一定的负性作用，而对排卵后黄体具有一定的正性作用。若能确定糖皮质激素的安全性，这一结果有望用于指导临床上糖皮质激素在辅助生殖中排卵后的支持治疗。