Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium.
Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium.
复制标题
无血清培养基中牛颗粒细胞增殖和黄体酮产生的控制因素。
DOI：
10.1210/endo-109-2-409
复制
发表时间：
1981
期刊：
影响因子：
4.8
通讯作者：
D. Gospodarowicz
中科院分区：
文献类型：
--
作者：
N. Savion;G. Lui;R. Laherty;D. Gospodarowicz
在有血清的情况下接种于细胞外基质包被培养皿的牛颗粒细胞，当暴露于添加胰岛素（2微克/毫升）、成纤维细胞生长因子（FGF，100纳克/毫升）和高密度脂蛋白（HDL，30微克蛋白质/毫升）的无血清培养基时会活跃增殖。培养物的最终密度是在添加最佳浓度（10%）小牛血清的培养基中生长的培养物的80 - 120%。当单独添加到无血清培养基中时，胰岛素对细胞增殖的影响最大，因为它使细胞数量增加，达到在最佳血清浓度下观察到的35 - 60%。当单独添加时，生长调节素C可以替代胰岛素。FGF、表皮生长因子或HDL本身对细胞增殖没有显著影响。当这些因子与胰岛素一起添加时，它们在刺激细胞增殖方面具有协同作用。当在完全无血清的情况下接种培养物时，为了使胰岛素和FGF具有促有丝分裂作用，需要在无血清培养基中添加转铁蛋白（10微克/毫升）。在细胞外基质上维持并仅暴露于无血清培养基的培养物在培养中的寿命为4代。添加胰岛素、FGF和HDL可将培养物的寿命增加到12代。在特定培养基中增殖的牛颗粒细胞通过向培养基中释放孕酮对二丁酰环腺苷酸作出反应。向特定培养基中添加促卵泡激素（FSH）导致细胞增殖减少30%，并且在对二丁酰环腺苷酸作出反应时向培养基中释放的孕酮量增加2.1倍。这种孕酮的释放达到与在添加最佳浓度血清的培养基中生长并且在其生长阶段和汇合时暴露或未暴露于FSH的培养物所观察到的相似水平。这些结果表明，牛颗粒细胞可以在无血清培养基中活跃增殖并维持其分化功能，这从它们产生孕酮的能力可以看出。
Bovine granulosa cells seeded in the presence of serum on extracellular matrix-coated dishes proliferate actively when exposed to serum-free medium supplemented with insulin (2 microgram/ml), fibroblast growth factor (FGF, 100 ng/ml), and high density lipoprotein (HDL, 30 microgram protein/ml). The final density of the cultures is 80-120% that of cultures grown in the presence of medium supplemented with optimal concentration (10%) of calf serum. Insulin has the greatest effect on cell proliferation when added alone to serum-free medium, since it induced an increase in cell number that was 35-60% that observed with optimal serum concentration. Somatomedin C can replace insulin when added alone. FGF, epidermal growth factor, or HDL had no significant effect on cell proliferation by themselves. When these factors were added together with insulin, they acted synergistically in stimulating cell proliferation. When cultures were seeded in the total absence of serum, the addition of transferrin (10 microgram/ml) to serum-free medium was required in order for insulin and FGF to be mitogenic. Cultures maintained on extracellular matrix and exposed to serum-free medium alone have a lifespan in culture of 4 generations. Addition of insulin, FGF, and HDL increases the lifespan of the cultures to 12 generations. Bovine granulosa cells, which proliferate in a defined medium, respond to dibutyryl cAMP by releasing progesterone into the medium. Addition of FSH to the defined medium resulted in a 30% decrease in cell proliferation and in a 2.1-fold increase in the amount of progesterone released into the medium in response to dibutyryl cAMP. This release of progesterone reached a level similar to that observed with cultures grown in medium supplemented with optimal concentration of serum and exposed or not to FSH during their growth phase and at confluence. These results demonstrate that bovine granulosa cells can actively proliferate in a serum-free medium and maintain their differentiated function, as indicated by their ability to produce progesterone.