经后增殖方含药血清对超排卵大鼠卵巢COCs及CCs中GDF9与Bim表达的影响

目的 观察经后增殖方含药血清对超排卵大鼠卵巢的卵丘-卵母细胞复合体(COCs)及卵丘细胞(CCs)中生长分化因子9(GDF9)及Bim表达的影响,探讨其治疗效果及作用机制.方法 制备大鼠超排卵卵巢模型,收集COCs和CCs,分别与雌性SD超排卵大鼠空白血清和经后增殖方药物血清在体外共同培养,各分3组:空白血清组(空白组)、含药血清组(对照组)、含药血清+ GDF9受体阻断剂组(实验组),共6组.24 h后收集COCs和CCs,实时荧光定量PCR检测GDF9和Bim mRNA表达水平,蛋白质印迹法检测GDF9蛋白表达水平.结果 (1)对照组COCs中GDF9 mRNA表达水平明显高于空白组和实验组(P＜0.05);对照组和实验组COCs中Bim mRNA表达水平均明显低于空白组(P＜0.05);对照组COCs中GDF9蛋白表达水平明显高于空白组(P＜0.05),亦高于实验组,但差异无统计学意义(P＞0.05).(2)CCs中GDF9 mRNA表达水平在3组间的差异性比较结果与COCs中一致;对照组和实验组CCs中Bim mRNA表达水平均明显低于空白组(P＜0.05);对照组CCs中Bim mRNA表达水平明显低于实验组(P＜0.05);对照组CCs中GDF9蛋白表达水平明显高于空白组和实验组(P＜0.05).(3)对照组COCs中GDF9 mRNA表达水平明显高于CCs中(P＜0.05),其余组内COSs和CCs比较差异无统计学意义(P＞0.05).结论 经后增殖方舍药血清能提高COCs和CCs中GDF9的表达水平,维持COCs和CCs中Bim的低水平表达,抑制细胞凋亡,促进卵泡发育;COCs较剔除卵母细胞的CCs更有利于GDF9 mRNA的表达.

Effect of serum containing Jinghou Zengzhi Recipe on expression of GDF9 and Bim in ovarian COCs and CCs in controlled ovarian hyperstimulation rats

Objective To observe the effects of serum containing Jinghou Zengzhi Recipe (JHZZ) on the expression of growth differentiation factor 9 (GDF9) and Bim in cumulus-oocyte complexes (COCs) and cumulus cells (CCs) of controlled ovarian hyperstimulation (COH) rats,and to investigate its curative effect and mechanism.Methods The rat COH ovarian model was prepared for collecting COCs and CCs,which were respectively co-cultured in vitro with the blank serum of female COH SD rat and serum containing JHZZ,and the each group was divided into 3 subgroups:the blank serum group (blank group),serum containing JHZZ group (control group) and serum containing JHZZ plus GDF9 receptor blocker group (experimental group),total 6 subgroups.COCs and CCs were collected after 24 h.The expression levels of GDF9 and Bim mRNA were detected by real-time quantitative PCR.The GDF9 protein expression levels were detected by Western blot.Results (1) The expression level of GDF9 mRNA in control group COCs was obviously higher than that in the control group and experiment group COCs (P＜0.05);the Bim mRNA expression level in COCs of control group and experiment group was significantly lower than that in COCs of blank group (P＜ 0.05);the GDF9 protein expression level in the control group COCs was significantly higher than that in the blank group COCs (P＜0.05),and also higher than that in the experiment group COCs,but the difference was not statistically significant (P＞0.05).(2)The comparison results of GDF9 mRNA expression level in CCs among 3 groups were consistent with those in COCs;the Bim mRNA expression level in CCs of control group and experimental group was significantly lower than that in the blank group (P＜ 0.05);the Bim mRNA expression level in the control group CCs was significantly lower than that in the experimental group CCs (P＜0.05);the GDF9 protein expression level in the control group CCs was significantly higher than that in the blank group and experimental group CCs (P＜0.05).(3)The GDF9 mRNA expression level in the control group COCs was significantly higher than that in CCs (P＜0.05),and the other inter-group comparisons between COSs and CCs had no statistical difference (P＞0.05).Conclusion Serum containing JHZZ can increase the GDF9 expression level in COCs and CCs,maintain the low expression of Bim in COCs and CCs,inhibit the ovarian cells apoptosis and promote the follicular development;COCs is more conducive to the expression of GDF9 mRNA compared with CCs eliminating oocyte.