【摘要】 背景:建立稳定、高效的颗粒细胞体外培养体系,可了解颗粒细胞体外生长的特点与基本生物学特性。目的:建立并优化小鼠卵巢颗粒细胞原代培养体系,探讨孕马血清促性腺激素、人绒毛膜促性腺激素对细胞增殖与细胞骨架的影响。方法:分离小鼠卵巢颗粒细胞,分别培养于RPMI1640、HTF与BIO-AMF2培养系统,将分离得到的第3代颗粒细胞培养体系中添加人绒毛膜促性腺激素与孕马血清促性腺激素进行干预。结果与结论:BIO-AMF2培养体系优于RPMI1640、HTF培养体系,原代培养第4天可见细胞汇合成片呈铺路石样生长,颗粒细胞C-kit免疫组化染色呈阳性,细胞周期测定表明体外培养的颗粒细胞大部分处于G1期。在颗粒细胞培养后第2天,激素对颗粒细胞的增殖即显现促进作用,在第4天最为显著,但未发现激素对颗粒细胞的细胞形态与细胞骨架有显著影响。更多还原

【Abstract】 BACKGROUND: The in vitro growth characteristics and basic biological characteristics of granulosa cells can be understood by building a stable and high-efficiency in vitro culture system of granulosa cells. OBJECTIVE: To build and optimize a primary culture system of mice ovarian granulosa cells, and to explore the effects of pregnant mare serum gonadotropin, human chorionic gonadotropin on the cell proliferation and cytoskeleton. METHODS: Mouse granulosa cells were separated and cultured in the culture systems of RPMI1640, HTF and BIO-AMF2, respectively. Pregnant mare serum gonadotropin and human chorionic gonadotropin were added to the culture systems of granulose cells in generation 3. RESULTS AND CONCLUSION: BIO-AMF2 culture system was better than RPMI1640 and HTF culture systems. Cells converged together and represented a cobblestone-like appearance on the 4th day after primary culture. The granulosa cells showed a positive reaction to C-kit immunohistochemical staining. According to cell cycle determination, most of the in vitro cultured granulosa cells were at G1 stage. Hormone showed a promoting effect on the proliferation of granulosa cells on the 2nd day after cultivation. This promoting effect was the most obvious on the 4th day after cultivation. But no significant effects of hormone on the granulosa cell morphology and cytoskeleton were detected.更多还原