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目的探讨补肾活血方含药血清对体外培养的大鼠卵巢颗粒细胞增殖分化及ActA/Smads信号传导通路的影响。方法采用机械分离法建立SD大鼠卵巢颗粒细胞体外培养体系。大鼠随机分为中药组,FSH组,胎牛血清组,无血清组。中药组再分为1%、5%、10%、15%、20%不同浓度含药血清培养液组,FSH组含FSH 25ng/ml,胎牛血清组含15%胎牛血清。ELISA检测E_2、P的分泌量。另设体外培养的颗粒细胞含药血清高、中、低剂量组,生理盐水组、FSH组、无血清组,空白组,分别于1h、2h、6h、12h、24h、48h后ELISA法测定ActA分泌量及免疫组化SP法测定ActRⅡA、Smad2、Smad3表达量。结果免疫组化FSHR阳性率大于95%。15%BSHX含药血清E_2分泌量最高,各组差异有统计学意义,P<0.05;各组P分泌量无显著差异,P>0.05。BSHX含药血清组ActA分泌量与时间存在一定的依赖性,12h后出现峰值,最佳剂量为中剂量组;各BSHX含药血清组中ActRⅡA、Smad2、Smad3免疫染色主要位于细胞核,且均与时间存在一定的依赖性,12h后出现峰值。结论 BSHX含药血清能促进体外培养的卵巢颗粒细胞增殖和分化,促进其E_2、P的分泌。通过促进ActA的分泌量和上调ActRⅡA、Smad2、Smad3表达来影响ActA/Smads信号通路的传导。
Objective To investigate the effects of Bushen Huoxue Recipe containing serum on the proliferation and differentiation of rat ovarian granulosa cells and the ActA / Smads signal transduction pathway. Methods SD rats ovarian granulosa cells culture system was established by mechanical separation. Rats were randomly divided into traditional Chinese medicine group, FSH group, fetal bovine serum group, serum-free group. The traditional Chinese medicine group was further divided into 1%, 5%, 10%, 15% and 20% different concentrations of serum-containing medium group, FSH group containing 25ng / ml FSH, fetal bovine serum containing 15% fetal bovine serum. ELISA detection E_2, P secretion. In addition, cultured in vitro granulosa cells containing serum high, medium and low dose group, saline group, FSH group, serum-free group, blank group, respectively, at 1h, 2h, 6h, 12h, 24h, Secretion and immunohistochemical SP method ActR Ⅱ A, Smad2, Smad3 expression. Results The positive rate of immunohistochemistry FSHR was more than 95%. The secretion of E_2 in 15% BSHX-containing serum was the highest, with statistical significance in all groups (P <0.05). There was no significant difference in P secretion between groups (P> 0.05). The concentration of ActA secreted in BSHX serum-containing group was dependent on the time, peaked at 12h, and the optimal dose was medium dose. The immunoreactive ActRⅡA, Smad2 and Smad3 in BSHX-containing serum were mainly located in the nucleus, There is a certain time dependence, peaked after 12h. Conclusion The serum containing BSHX can promote the proliferation and differentiation of ovarian granulosa cells in vitro and promote the secretion of E_2 and P. It can affect the conduction of ActA / Smads signal pathway by promoting the secretion of ActA and up-regulating the expression of ActRⅡA, Smad2 and Smad3.