重组蛋白GDF-9对卵巢黄素化颗粒细胞增殖和凋亡的影响

Effect of Recombinant Growth Differentiation Factor-9 on Cell Proliferation and Cell Apoptosis of Human Ovary Luteinized Granulosa Cell

目的探讨重组生长分化因子-9(GDF-9)对人卵巢黄素化颗粒细胞周期及细胞增殖、凋亡的影响。方法收集因输卵管和(或)男性因素而接受体外受精-胚胎移植(IVF-ET)治疗患者的卵泡液进行密度梯度离心,收集黄素化颗粒细胞,接种于M199培养基进行原代培养,实验组在M199培养基中添加100ng·m L-1GDF-9因子,对照组未添加GDF-9因子;培养48h后,用CCK-8试剂盒测定细胞增殖情况;用流式细胞仪测定细胞周期和细胞凋亡;Western blot法检测裂解的半胱氨酸蛋白水解酶3(cleaved-caspase-3)、细胞外信号调节激酶1/2(ERK1/2)及其磷酸化表达水平。结果原代培养的人黄素化颗粒细胞在培养第2~4天时处于分裂增殖高峰期,贴壁培养24h再添加GDF-9因子作用48h后,细胞周期检测结果显示,GDF-9使颗粒细胞周期表现为GO/Gl期的比例明显减少,S+G2/M期的比例明显增高(P<0.05)。细胞凋亡检测结果显示,颗粒细胞凋亡率显著减少(P<0.05)。Western blot检测发现,添加GDF-9使cleaved-caspase-3蛋白的相对表达量降低,磷酸化ERK1/2(p-ERK1/2)蛋白的相对表达量显著增高(P<0.05)。结论重组GDF-9因子可促进颗粒细胞增殖,抑制颗粒细胞凋亡,其机制可能与活化ERK和抑制cleaved-caspase-3途径、调节细胞周期有关。

Objective To explore the effect of recombinant growth differentiation factor-9( GDF-9) on the cell cycle,cell proliferation and apoptosis of luteinized granulosa cells from polycystic ovarian syndrome patients. Methods The primary luteinized granulosa cells collected from IVF-ET’s follicular fluid by density gradient centrifugation were cultured in M199 medium for 48 hours. Cell proliferation was measured by CCK-8 kit. Cell cycle and cell apoptosis were analyzed by flow cytometry. Western blot analysis was used to detect the expression level of cleaved-caspase-3 and Phosphorylated extracellular signal regulating kinase 1 /2( p-ERK1 /2). Results Primary granulosa cells cultured in vitro were in a split peak during the 2-5d. Exogenous GDF-9 could promote cell survival and proliferation may by regulating the G1-S cell cycle transition. The granulosa cells in S and G2 / M phase of cell cycle increased signficantly and G0 / G1 phase decreased signficantly( P < 0. 05). The apoptosis of the granulosa cells decreased signficantly after the treatment of GDF-9 for 48 h. Western Blot showed that the expression of cleaved-caspase 3 declined signficantly,ERK1 /2 and p-ERK1 /2 signficantly increased after the treatment of GDF-9 and the ERK activation rate increased significantly( P < 0. 05). Conclusion Exogenous GDF-9 promotes granulosa cell cycle progression and cell proliferation via activating ERK and inhibiting cleaved-caspase-3. It is required for normal follicular development and ovarian function restoration.