EGF促进绵羊附睾上皮细胞体外增殖的研究

EGF promoting proliferation of sheep epididymal epithelial cells in vitro

【目的】探讨表皮生长因子(EGF)对体外培养的绵羊附睾上皮细胞(EECs)增殖的影响。【方法】分离培养EECs,利用免疫荧光检测角蛋白18(CK18)对分离的细胞进行鉴定,用CCK-8法测定EGF的最佳作用质量浓度,用RT-PCR检测EECs中附睾分子标记——谷胱甘肽过氧化物酶-5(GPX5)和雄激素受体(AR)基因的表达情况,用流式细胞仪法检测EGF对EECs细胞周期和凋亡的影响。将P4代EECs分别用表皮生长因子受体(EGFR)抑制剂Gefitinib、磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路抑制剂LY294002和DMSO(对照组)处理后,再用EGF培养,用流式细胞仪法检测细胞周期和凋亡情况。将P4代EECs分别用EGF(-)、EGF、EGF+Gefitinib和EGF+LY294002处理后,用Western blot检测细胞中EGFR、AKT和叉头盒蛋白O1(FOXO1)磷酸化水平。【结果】分离培养的EECs可表达CK18,细胞纯度较好;EGF对EECs增殖促进效应呈浓度依赖性,用含50 ng/mL EGF培养基培养的EECs具有最高的增殖潜能,且不同传代EECs细胞均可正常表达GPX5和AR;EGF处理S期EECs细胞比例极显著升高(P<0.01),凋亡指数显著降低(P<0.05)。与对照组相比,Gefitinib组S期EECs细胞比例极显著降低(P<0.01),LY294002组S期细胞比例显著降低(P<0.05);Gefitinib组EECs细胞的凋亡指数极显著升高(P<0.01),而LY294002组EECs细胞的凋亡指数无显著变化。EGF组中EECs细胞的EGFR、AKT和FOXO1磷酸化水平较高;与EGF组相比,EGF+Gefitinib组中EECs细胞的EGFR、AKT和FOXO1磷酸化水平明显降低,而EGF+LY294002组中EECs细胞的AKT和FOXO1磷酸化水平明显降低。【结论】EGF可增强绵羊EECs体外生长活力,其作用机制可能是EGF通过介导EGFR和PI3K/AKT信号通路上调了FOXO1的磷酸化水平。

【Objective】The effects of epidermal growth factor(EGF)on sheep epididymal epithelial cells(EECs)cultured in vitro were studied.【Method】EECs were isolated and cultured.The isolated cells were identified by immunofluorescence detection of cytokeratin 18(CK18),and the optimal concentration of EGF was determined by CCK-8.The expression of epididymal molecular markers,glutathion peroxidase-5(GPX5)and androgen receptor(AR)genes,in EECs was detected by RT-PCR,and effects of EGF on cell cycle and apoptosis of EECs were detected by flow cytometry.After being treated with EGFR inhibitor(Gefitinib),PI3K/AKT inhibitor(LY294002)and DMSO(control group),the EECs at P4 were cultured with EGF and the cell cycle and apoptosis were detected by flow cytometry.The EECs at P4 were treated with EGF(-),EGF,EGF+Gefitinib and EGF+LY294002,and the phosphorylation levels of EGFR,AKT and FOXO1 were detected by Western blot.【Result】The isolated and cultured EECs expressed CK18 with good cell purity.EGF promoted proliferation of EECs in a concentration-dependent manner.EECs cultured in 50 ng/mL EGF medium had the highest proliferation potential,and GPX5 and AR could be expressed normally in EECs at different passages.The proportion of S phase EECs treated by EGF increased significantly(P<0.01),while the apoptotic index of EECs decreased significantly(P<0.05).Compared with the control group,the proportion of S phase EECs in Gefitinib group decreased extremely significantly(P<0.01),while the proportion of S phase EECs in LY294002 group decreased significantly(P<0.05).The apoptosis index of EECs treated with Gefitinib was significantly increased(P<0.01),while the apoptotic index of EECs treated with LY294002 had no significant difference.The phosphorylation levels of EGFR,AKT and FOXO1 in EECs cells treated with EGF were higher compared with EGF group,the phosphorylation levels of EGFR,AKT and FOXO1 in EECs of EGF+Gefitinib group were significantly decreased,and the phosphorylation levels of AKT and FOXO1 in EECs of EGF+LY294002 group were significantly decreased.【Conclusion】EGF can enhance the growth activity of sheep EECs in vitro,and the possible mechanism is that EGF up-regulates FOXO1 phosphorylation by inducing EGFR/PI3K/AKT pathway.