二甲双胍对体外培养人卵巢颗粒细胞分泌功能的影响

Effect of metformin on secretion of human luteinized granulosa cells in vitro

目的本研究探讨不同浓度二甲双胍在不同时间点对其分泌类固醇激素的影响。方法收集因输卵管因素不孕或者男性因素不孕行IVF/ICSI-ET患者促排卵后的含有黄素化颗粒细胞的卵泡液,经密度梯度离心后分离获得颗粒细胞,行体外培养。分别加入含有二甲双胍浓度为0 ng/ml,10 ng/ml,100ng/ml,1000ng/ml,10000ng/ml的DMEM培养基对其进行体外培养,每24h收集细胞培养上清,用化学发光法检测上清液中雌二醇及孕酮的水平。结果体外培养颗粒细胞分泌类固醇激素在72h到96h达到高峰,120h后开始递减,这与二甲双胍体外生长曲线一致;二甲双胍都能够抑制类固醇激素的分泌,且二甲双胍浓度在10000ng/ml,能够极显著抑制类固醇激素的分泌,在每个时间点均呈剂量依赖性。结论二甲双胍在颗粒细胞体外发育整个过程中均抑制。

Objective ： To investigate the direct effect of mefformin on ovarian steroidogenesis of granulose cells at different time. Methods： Human luteinized granulosa cells were isolated from patient during in vitro fertilization or intraeytoplasmie sperm injection under Density gradient centrifugation and cultured in the absence or presence of mefformin at different concentration. After treated with mefformin at different concentration for every 24h, cell proliferation was studied by measuring the OD and culture medium was collect- ed Estradiol and Progesterone production was determined by chemi - luminescence immunoassay per 24h. Results ： Steroid reached the peak during 72h to 96h and came down at 120h, which is in consistent with the growth curve. In vitro, metformin can inhibit the secretion of steroid. E2 and P was significantly inhibited at the concentration of 10000ng/ml. And there was a clear, dose - inde- pendent inhibition of E2 and P production by mefformin. Conlusion： mefformin inhibit the steroidogensis during the whole culture in vitro.