表皮生长因子对卵巢黄素化颗粒细胞cyclin D1和P21表达的影响  

Effects of Epidermal Growth Factor on the Expression of Cyclin D1 and P21 in Ovarian Luteinized Granulosa Cells

作　　者：李雪[1] 裴承斌 杨玲玲 何艳桃 侯巧妮 赵帅[1] 虎娜 马会明[1] LI Xue;PEI Chengbin;YANG Lingling;HE Yantao;HOU Qiaoni;ZHAO Shuai;HU Na;MA Huiming(Key laboratory of Fertility Preservation and Maintenance of Ministry of Education,Ningxia Medical University,Yinchuan 750004,China;Ningxia Human Spermbank,the General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学生育力保持教育部重点实验室,银川750004 [2]宁夏医科大学总医院宁夏人类精子库,银川750004

出　　处：《宁夏医科大学学报》2020年第5期440-444,462,共6页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81660813);宁夏高等学校科学研究项目(NGY2017130)。

摘　　要：目的探讨表皮生长因子(EGF)对颗粒细胞细胞周期的影响及其在颗粒细胞增殖、分化机制中的作用。方法选取IVF-ET助孕方式治疗患者的卵巢黄素化颗粒细胞为研究对象,分离纯化后分为对照组和实验组,对照组为正常培养的颗粒细胞,不添加EGF;实验组为添加10 ng·mL^-1EGF培养24 h的颗粒细胞。利用CCK-8试剂盒在450 nm波长条件下用酶标仪检测细胞增殖状况以观察EGF作用的最佳浓度;利用流式细胞仪检测EGF对颗粒细胞周期的影响;利用RT PCR技术检测EGF对颗粒细胞cyclin D1和P21 mRNA相对表达的影响;利用Western blot技术检测EGF对颗粒细胞cyclin D1和P21相对蛋白表达水平的影响。结果采用本研究所建立的方法可以获得生长活力好、细胞纯度高以及较稳定的人卵巢黄素化颗粒细胞;CCK-8结果显示EGF最佳作用浓度为10 ng·mL^-1;细胞周期结果显示,EGF使人卵巢黄素化颗粒细胞周期表现为G0/G1期的比例减少,S+G2/M期的比例增高(P均<0.05);RT PCR结果显示,与对照组相比EGF作用后cyclin D1基因相对表达量升高,P21基因相对表达量下降(P均<0.01);Western blot结果显示,与对照组相比EGF作用后,颗粒细胞cyclin D1蛋白相对表达量升高,P21蛋白相对表达量降低(P均<0.05)。结论添加10 ng·mL^-1浓度的EGF培养人卵巢颗粒细胞可通过对细胞周期调控因子cyclin D1和P21mRNA及蛋白表达水平的影响,促进颗粒细胞增殖。Objective To investigate the effect of epidermal growth factor(EGF)on the cell cycle of granulosa cells and its role in the mechanism of proliferation and differentiation of granulosa cells Methods The luteinized granulosa cells from the ovaries of patients treated with IVF-ET assisted pregnancy were selected as the research objects.After separation and purification,they were divided into the control group and the experimental group.The control group was normal cultured granulosa cells without EGF and the experimental group was added with 10 ng·mL^-1 EGF for 24 hours.The CCK-8 kit was used to detect the cell proliferation and the optimal concentration of EGF under the wavelength of 450 nm.The flow cytometry was used to detect the effect of EGF on the cell cycle of granulosa cells.RT PCR technology was used to detect the effect of EGF on the relative mRNA expression of cyclin D1 and P21 of granulosa cells.Western blot was used to detect the effect of EGF on protein expression of cyclin D1 and P21 in granulosa cells.Results Human ovarian luteinized granulosa cells with good growth activity,high cell purity and stability can be obtained by using the method established in this study.CCK-8 results showed that the optimal EGF concentration was 10ng·mL^-1;cell cycle results showed that the human ovarian iuteinized in S and G2/M phase of cell cycle increased signficantly and G0/G1 phase decreased signficantly(P<0.05);RT PCR results showed that compared with control group EGF increased the relative expression of cyclin D1 gene and decreased the relative expression of P21 gene(P<0.01);Western blot results showed that compared with control group the relative expression of cyclin D1 protein increased and P21 protein decreased after EGF treatment(P<0.05).Conclusion Human ovarian granulosa cells were cultured 24 h with 10 ng·mL^-1 EGF can enhance the proliferation ability of human ovarian granulosa cells by impacting the protein and gene expression of cyclin D1 and P21.

关 键 词：表皮生长因子 黄素化颗粒细胞 细胞周期 cyclin D1 P21 

分 类 号：R711.75[医药卫生—妇产科学]