Effects of basal media and supplements on diethylstilbestrol-treated immature mouse primary granulosa cell growth and regulation of steroidogenesis in vitro.
Effects of basal media and supplements on diethylstilbestrol-treated immature mouse primary granulosa cell growth and regulation of steroidogenesis in vitro.
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DOI：
10.1111/j.1439-0531.2011.01879.x
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发表时间：
2012-06
期刊：
影响因子：
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通讯作者：
T. Ding;A. Luo;S. Yang;Z. Lai;Y. Wang;W. Shen;J. Jiang;Y. Lu;D. Ma;S. Wang
中科院分区：
文献类型：
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作者：
T. Ding;A. Luo;S. Yang;Z. Lai;Y. Wang;W. Shen;J. Jiang;Y. Lu;D. Ma;S. Wang
本研究的目的是研究基础培养基、补充物（包括5 mg/ml胰岛素、5 mg/ml转铁蛋白和5 μ g/ml硒的混合物（ITS））、牛血清白蛋白（BSA）、丙酮酸钠（PNa）和表皮生长因子（EGF）与血清对己烯雌酚处理的C57 BL/6 J小鼠原代颗粒细胞（GC）增殖的影响进行比较，游离培养体系，并利用该体系研究这些补充剂对GCs类固醇合成过程的影响及相关调控机制。选择DMEM/F_（12）、麦考伊s 5a、RPMI Medium 1640、Medium 199和α-MEM作为基础培养基，其中麦考伊s 5a对GCs增殖有显著促进作用（p < 0.05），且附着力和形态学均较好。颗粒细胞在含血清培养液中的增殖状况优于无血清培养液，BSA、ITS、PNa和EGF对无血清培养液中颗粒细胞的增殖有促进作用，而后三者对血清培养液中颗粒细胞的增殖有相同的促进作用。培养2天后，培养的细胞保持P450 arom阳性，其维持正常形态，并且当在无血清条件下添加补充剂时没有凋亡迹象。只有ITS诱导雌二醇的产生，而ITS，BSA和PNa促进孕酮分泌显着表示促进分化。这些补充剂的作用部分是通过提高星星、3β-HSD、P450 SCC和P450 arom的转录水平来实现的。结论：麦考伊5a培养基是较好的基础培养基，ITS和PNa对原代GCs的增殖和分化均有促进作用，为GCs的生理研究提供了较好的条件。
The objectives of this study were to investigate the effects of basal media, supplements, including mixture of 5 mg/ml insulin, 5 mg/ml transferrin and 5 g/ml selenium (ITS), bovine serum albumin (BSA), sodium pyruvate (PNa) and epidermal growth factor (EGF) compared with serum on the proliferation of diethylstilbestrol-treated C57BL/6J mouse primary granulosa cells (GCs) to develop a serum-free culture system and to use this system to evaluate the effects of these supplements on GCs' processes of steroidogenesis and related regulation mechanism. DMEM/F12, McCoy's 5a, RPMI Medium 1640, Medium 199 and a-MEM were chosen as basal media, and McCoy's 5a proliferated the GCs greatly (p < 0.05) with a better attachment and morphology. Granulosa cells showed a better proliferation status in serum-containing media than serum-free media, BSA, ITS, PNa and EGF increased the proliferation of GCs in serum-free system gradually, while the latter three had the same effect on supporting GCs' proliferation with serum. After 2-day culture, the cells cultured remained P450arom positive, which sustained a normal morphology and there was no sign of apoptosis when supplements were added in the serum-free condition. Only ITS induced oestradiol production, while ITS, BSA and PNa enhanced progesterone secretion significantly indicating the promotion of differentiation. And these supplements functioned partly through increasing the transcription level of StAR, 3β-HSD, P450scc or P450arom. In conclusion, McCoy's 5a medium was a better basal medium, and ITS and PNa promoted both the proliferation and differentiation of primary GCs in serum-free culture system, showing a satisfactory state for GCs' physiological study.