Bcl-2基因RNA干扰对体外培养的鹅卵泡颗粒细胞凋亡、增殖和孕激素分泌的影响

Effects of Bcl-2 Gene Interference on the Apoptosis,Proliferation and Progesterone Secretion of Goose Follicular Granulosa Cells

根据已发表的Bcl-2基因序列设计3对siRNA（Small interference RNA）序列，构建干扰表达载体，转染体外培养的鹅卵泡颗粒细胞，48h后利用流式细胞术（Flow cytometry，FCM）检测颗粒细胞Bcl-2蛋白表达量、凋亡和增殖情况，收集细胞培养液用放射免疫分析法检测孕激素（P）的分泌水平；此外，还检测了F1～F4卵泡、最小的排卵前卵泡（The smallest preovulatory follicle，SPF）、小黄卵泡（Small yellow follicle，SYF）和闭锁卵泡（Atresic follicle）颗粒细胞Bcl-2蛋白表达和凋亡情况。结果表明：（1）各级卵泡颗粒细胞Bcl-2蛋白的表达存在着差异，正常卵泡颗粒细胞Bcl-2蛋白表达量显著高于闭锁卵泡（P〈0．05），SPF颗粒细胞Bcl-2蛋白表达水平显著高于SYF（P〈0．05）；（2）干扰组Bcl-2蛋白表达水平显著低于所有对照组（P〈0．05），而细胞凋亡指数（Apoptosis index，AI）、增殖指数（Proliferation index，PI）和P分泌水平均高于对照组。

According to the published sequences, three siRNAs （Small interference RNA） were designed, and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro. The expression level of Bcl-2 protein, apoptosis and proliferation of granulosa cells were analyzed by flow cytometry 48 h after the transfection, and the progesterone （P） in the medium was assayed using RIA. Meanwhile, apoptosis indexes and Bcl-2 protein level of follicular granulosa cells with different rank were also analyzed. Our results indicated that： （1） Levels of Bcl-2 protein in follicular granulosa cells with different rank were different, the highest expression level was observed in the smallest preovulatory follicle （SPY）, and levels of Bcl-2 protein in the healthy follicles were significant higher than that in atresic follicles （P 〈 0.05） ; （2） Bcl-2 protein levels of test groups were significant lower than that of control （P 〈 0.05）, while apoptosis indexes （AI）, proliferation indexes （PI） and P secretion of test groups were higher than that of control （P 〈0.05）.