BDNF/TrkB 信号通路对牛卵泡颗粒细胞类固醇激素分泌的影响及机制研究

Granulosa cells play important roles in follicular development. It has been shown that BDNF/TrkB pathway is closely related to the initiation and maintainnence of follicle deveolpment as well as development and apoptosis of oocytes. However, there is still no data availeble on the relationship between the BDNF/TrkB pathway and synthesis and secretion of steroids in granulosa cells. This projects will firstly detect the expression and distribution of BDNF and TrkB in bovine ovaries, then, by establishing in vitro model of granulosa cells for the inhibition and activation of BDNF/TrkB pathway or supplementation of BDNF in culture media, explore the effects of BDNF/TrkB pathway on secretion of steroids. The project will also study the changes of key enzymes for steroids synthesis and related signaling pathways to reveal the mechanism of action of BDNF on bovine granulosa cells.

卵泡颗粒细胞是决定卵泡状态的重要细胞。业已清晰BDNF/TrkB信号通路与卵泡发育的启动与维持、卵母细胞的发育与凋亡密切相关，但与颗粒细胞类固醇激素分泌功能的关系至今尚不清楚。本项目首先检测BDNF与TrkB在牛卵巢中的分布，在此基础上运用颗粒细胞体外培养体系，通过信号通路阻抑细胞模型和添加外源性BDNF等方法，分析确定该通路对体外培养牛卵泡颗粒细胞激素分泌的影响；通过检测激素合成关键酶与相关信号通路的变化，探析BDNF/TrkB通路影响卵泡颗粒细胞激素分泌功能的机制，为阐明BDNF/TrkB通路在卵泡发育中的作用提供新的理论依据。

本项目研究了BDNF/TrkB信号通路对牛卵泡颗粒细胞类固醇激素（E2，P）分泌的影响。首先，建立牛卵泡颗粒细胞体外培养体系，运用细胞免疫荧光技术检测了BDNF与TrkB在牛卵泡颗粒细胞中的表达。然后，利用外源添加BDNF以及RNAi和K252a阻抑的方法处理颗粒细胞，测定上清中E2和P的浓度；同时采用Real-time PCR技术和Western blotting方法检测类固醇合成过程中的关键限速酶P450scc、P450arom和StAR的表达，结果表明，BDNF和TrkB在牛卵泡颗粒细胞上均有表达；当外源添加100ng/ml的BDNF时，颗粒细胞对E2和P的分泌较对照组相比有显著性升高；相应地，P450scc、P450arom和StAR的表达均呈上调趋势；利用si-BDNF和100ng/ml的K252a阻抑BDNF/TrkB信号通路时，E2和P的分泌则降低，与之相对应地，P450scc、P450arom和StAR的表达也呈下降趋势。以上实验结果初步探明BDNF/TrkB信号通路参与了对牛卵巢颗粒细胞类固醇激素E2和P分泌的调控，这种调控作用是通过调节激素合成限速酶实现的。