安徽医科大学硕士学位论文慢性应激对卵泡发育的影响及自分泌、旁分泌调控机制研究姓名：沈妮申请学位级别：硕士专业：指导教师：刘雨生20100101安徽医科大学硕士学位论文 4 慢性应激对卵泡发育的影响及自分泌、旁分泌调控机制研究 慢性应激对卵泡发育的影响及自分泌、旁分泌调控机制研究 摘 要 目的： 观察慢性应激对卵泡发育和动情周期的影响及其对卵泡发育的非促性腺激素依赖的影响。选取 GDF-9、BMP-15、bFGF、SCF 四个代表性的卵巢旁分泌、自分泌因子行 RT-PCR，观察慢性应激是否影响这些调控卵泡发育的卵巢局部调控因子的表达。 方法： （1）通过建立小鼠慢性应激模型，制备卵巢 HE 切片观察慢性应激对卵泡发育的影响。 （2）从慢性应激的第一天开始（至处死日止）取小鼠阴道分泌物，作涂片，行瑞氏染色观察其所处动情周期。 （3）通过外源性补充促性腺激素，观察其对应激后卵泡发育的恢复作用。 （4）采用相对定量 real-time PCR 方法测定 GDF-9、BMP-15、bFGF、SCF 这几个代表性的卵巢旁分泌、自分泌因子的 mRNA 水平。 实验结果： （1）慢性应激使小鼠的动情周期紊乱、间期延长。 （2）慢性应激影响卵泡的发育及排卵的发生。 （3）外源性补充促性腺激素不能使卵泡发育和排卵完全恢复正常。 （4）卵源性自分泌、旁分泌因子GDF-9、BMP-15的mRNA表达水平有明显下降，而颗粒细胞源性自分泌、旁分泌因子SCF、bFGF的mRNA表达水平则无变化。 安徽医科大学硕士学位论文 5 结论： 慢性应激可以影响小鼠的动情周期，影响小鼠卵泡的发育及排卵。外源性补充促性腺激素后，慢性应激组排卵数明显减少，HE染色结果显示：慢性应激增加了卵泡的闭锁，干扰了卵泡的发育和排卵，即使外源性给予促性腺激素仍不能使早期卵泡发育和排卵恢复正常，提示非促性腺激素类因素参与慢性应激干扰卵泡发育和排卵。RT-PCR结果显示，慢性应激后，相比正常对照组，自分泌、旁分泌因子GDF-9、 BMP-15表达水平有明显降低 （P0.05） ， SCF、 bFGF表达无明显变化。提示慢性应激通过下调卵源性的局部旁分泌因子GDF-9、BMP-15参与影响卵泡的发育。 关键词：慢性应激；自分泌、旁分泌因子；卵泡；发育 The effect of Chronic stress on follicular development and 安徽医科大学硕士学位论文 6 mechanism of autocrine, paracrine regulatory factors Abstract Objective Observe the effects of chronic stress on follicular growth and the estrous and the effect on the follicle development of non-gonadotropin-dependented .Then checkout four paracrine-autocrine factors for RT-PCR,and see if they participate in the process of chronic stress on follicular growth and the development of a disincentive effect. Methods (1) Through the establishment of chronic stress model in mice ,and prepare HE sections to observe the impaction of the growth and development of ovarian follicles. (2) From the first day of chronic stress(to execute ended),mice were vaginal discharge for smear,Wright strain to observe the line of estrous cycle. (3) Added by exogenous gonadotropin to observe their effection of recovery on ovulation after chronic stress. (4) Using real-time PCR semi-quantitative experimental to determine the self-secreted paracrine factors which were selected. Results (1) Chronic stress extend and disorder the sexual cycle of mice . (2)Chronic stress Inhibite follicle maturation and ovulation occurring 安徽医科大学硕士学位论文 7 (3) Added exogenous gonadotropin can not make the follicular development and ovulation back to normal completely. (4) In the course of chronic stress, the secretion of paracrine factor GDF-9、BMP-15 from oocyte make a evidence reduction in the level of mRNA and SCF、 bFGF from granulosa cells did not change. Conclusion Chronic stress can affect the estrous cycle in mice, affecting mouse follicle development and ovulation. Added exogenous gonadotropin, the chronic stress group significantly reduced the number of ovulation, HE staining showed: Chronic stress increased follicle atresia, interferes with follicular development and ovulation, even if add exogenous gonadotropin still can not make the early follicular development and ovulation back to normal, suggesting that the non-gonadotropin-class factors are involved in chronic stress interfere with follicular development and ovulation. RT-PCR results showed that after chronic stress, since the secretion of paracrine factor GDF-9、 BMP-15 make a evidence reduction, SCF, bFGF did not change,suggest that chronic stress pass the way of down regulation the secretion of paracrine factors GDF-9、BMP-15, these factors may impact follicle development . Keywords Chronic stress / autocrine,paracrine-factors/follicle development 安徽医科大学硕士学位论文 2 英文缩略词表（abbreviation） 英文缩写 英文名 中文名 HE Hematoxylin and Erosin 苏木精和伊红 DEPC Diethyl pyrocarbonate 焦炭酸二乙酯 mRNA Messengerribonucleicacid 信使核糖核酸 RT-PCR Reverse transcription-polymerase chain reaction 逆转录-聚合酶链反应 GDF-9 Growth differentiation factor 9 生长分化因子9 BMP-15 Bone morphogenetic p rotein 15 骨形态发生蛋白- 15 bFGF Basic fibroblast growth factor 碱性成纤维细胞生长因子 SCF Stem cell factor 干细胞因子 HPA Hypothalamus-pituitary-Adrenals 下丘脑-垂体-肾上腺轴 HPG Hypothalamus-pituitary-gonad 下丘脑-垂体-性腺 CRH Corticotropin-releasing factor 下丘脑促肾上腺皮质激 素释放激素 ACTH Adrenocorticotrophichormonepituitarygland 垂体促肾上腺皮质激素GC Glucocorticosteroid 肾上腺糖皮质激素 GnRH Gonadotrophin releasing hormone 下丘脑促性腺激素释放 激素 FSH Follicule-stimulating hormone 卵泡刺激素 LH Luteotropic hormone 黄体生成素 E2 Estradiol 雌二醇 LHR Luteotropic hormone receptor 黄体生成素受体 安徽医科大学硕士学位论文 3 FGFR Fibroblast growth factor receptor 纤维母细胞生长因子受体 PMSG Pregnant mare serum gonadotropin 孕马血清促性腺激素 HCG Human chorionic gonadotropin 人绒毛膜促性腺激素 Real-time PCR Nitric oxide synthase Real-time聚合酶链式反 应 Gn Gonadotrophin 促性腺激素 FSHR Follicle stimulating hormone receptor 卵泡刺激素受体 PA Plasminogen activator 纤溶酶原激活剂 安徽医科大学硕士学位论文 8 慢性应激对卵泡发育的影响及自分泌、旁分泌调控机制研究 慢性应激对卵泡发育的影响及自分泌、旁分泌调控机制研究 1. 前言 应激(stress)是指机体对内外环境剧变的刺激作出包括精神、 神经内分泌和免疫等方面的综合应答状态。现代社会竞争日益加剧，工作和生活节奏的不断加快，长期的压力使现代人处于慢性应激状态。2004年底中国科学院心理研究所历时4年完成的中国科学院知识创新工程项目子课题“社会转型期不同职业群体主要社会应激源与心理健康研究”的统计数据显示，近70%的受调查者感觉到压力的困扰，女性生殖系统对慢性应激尤为敏感，不孕、月经失调、闭经、多囊卵巢综合征等女性常见生殖系统疾病均与身心应激密切相关1。 慢性应激使得HPA轴处于持续激活状态，表现为CRH、ACTH和GC分泌持续性增加。 HPA轴的过