新加归肾丸通过调节mTOR信号通路促进体外培养大鼠卵巢颗粒细胞增殖

目的:观察补肾活血中药复方新加归肾丸对体外培养大鼠卵巢颗粒细胞增殖的影响。方法:体外培养大鼠卵巢颗粒细胞,分为空白组,模型组,中药低、中、高剂量组共五组,空白组用1%胎牛血清培养基培养,其余四组滴加雷帕霉素,20 min后中药各组加入不同浓度的中药水提醇沉液。分别在培养24、48、72 h后采用流式细胞仪检测细胞周期各时相的变化,并计算细胞增殖指数(PI); 免疫印迹法检测磷酸化-哺乳动物雷帕霉素靶蛋白(p-mTOR)、细胞周期蛋白D₂(Cyclin D₂)的表达水平。结果:用药24、48、72 h 这3个时段,中药各组S期细胞比率、PI均高于模型组(P<0.05),其中中药中剂量组最为明显(P<0.05)。用药24、48、72 h这3个时段,中药各个剂量组p-mTOR、Cyclin D₂表达水平均高于模型组(P<0.05)。中药各个剂量中以中药中剂量组效果最明显(P<0.05)。结论:补肾活血中药复方新加归肾丸能通过激活mTOR信号通路活性,上调Cyclin D₂蛋白表达,促使颗粒细胞由G₁期向S期转化,加速卵巢颗粒细胞周期进程,促进颗粒细胞增殖来实现改善卵泡发育。

Study on therapy of Xinjia Guishenwan of granulosa cells isolated from rat ovary and ecultured in vitro by regulating mTOR signal pathway

Objective:To study the effect of Xinjia Guishenwan,compund Chinese medcine with the function of Bushen Huoxue,on proliferation of granulosa cells isolated from rat ovary and ecultured in vitro.Methods:Cultured granulosa cells isolated from rat ovary in vitro and divided them into blank groupmodel group,high-dosage TCM medicine group,mid-dosage TCM group,low-dosage TCM group.The blank group was cultured in 1% fetal bovine serum medium,the rest 4 groups were dripped with rapamycin for 20 minutes,then added water extracting-alcoholprecipitating solution of traditional Chinese medicine with different concentrations into TCM groups.After 24,48 and 72 hours of culture,detected the changes of each phase of granule cell cycle in each group by flow cytometry,and calculated the proliferation index(PI).The expression of p-mTOR、Cyclin D₂ were detected by wstern blot.Results:At the 3 time periods(24 h,48 h,72 h)after therepy,the S-phase cells ratio and PI index of the TCM groups were all signifi-cantly higher than those of the model group(P<0.05).Among three TCM groups,the mid-dosage TCM group has the most obvious effect.At the 3 time periods(24 h,48 h,72 h)after therepy,the expression of mTOR and Cyclin D₂ protein in the TCM groups were all significantly higher than those of the model group(P<0.05).The most evident effect was the mid-dosage TCM group.Conclusion:Compound Chinese medcine Xinjia Guishenwan for Bushen Huoxue improving can improve follicular development,maybe this effect is achieved by activating the activity of mTOR signal pathway and up-regulating the expression of Cyclin D₂,induced the granulosa cell from G₁ to S period,then accelerate cell cycle period and promote the prolifer-ation of granulosa cells.