人卵巢颗粒细胞分离培养的优化及生物学特性

目的:建立原代人卵巢颗粒细胞(hGCs)的分离培养方法,观察其生物学特性。方法:取卵日,收集hGCs,分为对照组(DMEM培养基)和实验组(DMEM培养基∶卵泡液=1∶1)进行培养。比较2组hGCs的生长状态和雌激素分泌情况。利用RT-qPCR检测Nanog、Oct4、Sox2的表达,应用流式细胞术检测CD44、CD73、CD45的表达。结果:hGCs培养24 h贴壁良好,细胞间伸出伪足。对照组和实验组分别在培养14 d和21 d后出现凋亡。实验组雌激素的分泌显著增加,第9天出现高峰,第21天显著下降。培养第7天实验组Oct4和Nanog mRNA表达显著增加。培养7 d后,CD44、CD73表达阳性, CD45表达阴性。结论:该方法简单、有效,可获得大量hGCs。培养基中添加卵泡液有益于hGCs的生长,增加培养天数。同时hGCs可分泌雌激素并具有一定的干细胞特性。

Culture technique optimization and biological characteristics of human ovarian granulosa cells

Objective : To establish the methods of isolating and culturing human ovarian granulosa cells(hGCs) and observe biological characteristics. Methods : hGCs were harvested during oocyte retrieval. Then they were divided into control group(DMEM medium) and experimental group(DMEM medium : follicular fluid=1∶1). The characteristic of hGCs and secretion of estrogen were observed. The expression levels of Nanog, Oct4 and Sox2 were detected by RTqPCR. CD44, CD73 and CD45 were measured via flow cytometry(FCM). Results : The hGCs grew well in vitro after24 h, and cells were connected by pseudopodia. Apoptosis began in the cells of control group after 14 d, while cells of experimental group began to degenerate until 21 d. Compared with control group, the secretion of estrogen in the experimental group increased significantly. It reached the peak at 9 d and significantly declined at 21 d. The results from RT-qPCR indicated that the expression levels of Oct4 and Nanog of experimental group increased significantly at 7 d.FCM analysis showed that CD44 and CD73 were positive and CD45 was negative after 7 d in vitro culture. Conclusion : This method is simple and effective to obtain a large number of hGCs. Compared with the culture medium, adding follicular fluid is beneficial to the growth of hGCs, and increase the culture time. Meanwhile, hGCs can secrete estrogen and has certain stem cell characteristics.