激活素A调节人卵巢颗粒细胞Smad2和CYP19A表达的研究

Research of activin A on modulation of the expression of Smad2 and CYP19A in human ovarian granulosa cells

目的 探讨激活素A通过Smad2和CYP19A表达促进颗粒细胞增殖的作用途径。方法2017年3~8月,在宁夏医科大学总医院生殖医学中心就诊的患者中,选取体外受精-胚胎移植(IVFET)或卵胞质内单精子注射(ICSI)两种助孕方式治疗的15例患者为研究对象。取分离纯化的卵巢颗粒细胞,经原代培养48h后分组:激活素A组添加50μg/ml的激活素A,Bimagrumab组添加5μM选择性ActRⅡ抑制剂Bimagrumab,混合组同时添加50μg/ml的激活素A与20μM的Bimagrumab,再继续培养24h。采用Western印迹法检测蛋白表达水平,逆转录及实时PCR检测mRNA表达水平,细胞免疫荧光化学检测蛋白定位。采用单因素方差分析、t检验进行统计学分析。结果 激活素A组中ActRⅡ、p-Smad2/Smad2蛋白表达水平均高于空白对照组,Bimagrumab组中ActRⅡ和p-Smad2/Smad2蛋白表达水平低于空白对照组,差异有统计学意义(P<0.05),提示Bimagrumab对ActRⅡ有抑制效果,激活素A对ActRⅡ有促进效果。混合组中CYP19A1蛋白表达低于空白对照组,激活素A组中CYP19A1蛋白表达水平高于空白对照组;差异有统计学意义(P<0.05)。激活素A组中Smad2和CYP19A1mRNA表达水平高于空白对照组,混合组中Smad2和CYP19A1mRNA表达水平低于空白对照组,差异均有统计学意义(P<0.05)。细胞免疫化学染色显示,Smad2和CYP19A1存在于卵巢颗粒细胞中,Smad2阳性染色定位于细胞胞浆,CYP19A1阳性染色定位于细胞胞浆和细胞膜。结论 颗粒细胞分泌因子激活素A激活ActRⅡ信号通路,增加p-Smad2/Smad2和CYP19A1的表达水平,进一步促进颗粒细胞增殖和分化。

Objective To explore the effect of activin A to promote the proliferation of granulosa cells through the expression of Smad2 and CYP19A. Methods From March to August 2017, 15 cases were included who were treated by in vitro fertilization and embryo transfer(IVF-ET) and intracytoplasmic sperm injection (ICSI) in the center of reproductive medicine of General Hospital of Ningxia Medical University. The granulosa cells with isolating from follicular fluid were selected as the research object. After incubation for 48 hours, 50 μg / ml activin A was added to activin A group, 5 μM selective ActR Bimagrumab Ⅱ inhibitors was added to Bimagrumab group, 50 μg / ml activin A and 20 μM Bimagrumab were added to hybrid group, all of those were incubated for 24 h. The protein expression level was detected by Western blot assay. The mRNA expression level was detected by reverse transcription and real-time PCR. The protein localization was detected by immune-fluorescence cytochemistry technology. One-Way ANOVA and t test were used for statistical analysis. Results The protein expression level of ActR Ⅱ, p-Smad2/Smad2 in activin A group was higher than that in blank control group, and the protein expression level of ActR Ⅱ, p-Smad2/Smad2 in Bimagrumab group was lower than that in blank control group, which was statistical significance (P<0.05). It showed Bimagrumab has inhibited effect for ActR Ⅱ and activin A has promotional effect for ActR Ⅱ. The protein expression of CYP19A1 in hybrid group was lower than that in blank control group , the protein expression level of CYP19A1 in activin A group was higher than that in blank control group, which was statistical significance (P<0.05).The expression level of Smad2 and CYP19A1 mRNA in activin A group was higher than that in blank control group. The expression level of Smad2 and CYP19A1 mRNA in hybrid group was lower than that in blank control group. There were all statistical significance (P<0.05). Immunocytochemical staining technique showed Smad2 and CYP19A1 existed in granulosa cells of ovary, positive staining of Smad 2 was located in cytoplasm, positive staining of CYP19A1 was located in cytoplasm and cell membranes. Conclusion Activin A of granulosa cells can activate signaling pathways of ActR Ⅱ and improve the expression level of p-Smad2/Smad2 and CYP19A1, which can further enhance the proliferation, and differentiation of granulosa cells.