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中电导钙激活钾通道调节表皮生长因子诱导的人脐静脉内皮细胞体外增殖
Intermediate conductance Ca2+ activated K+ channels 3.1 regulates the proliferation of human umbilical vein endothelial cells induced by epidermal growth factor in vitro
目的 观察表皮生长因子（EGF）对人脐静脉内皮细胞（HUVECs）离子通道中电导钙激活钾通道（KCa 3.1）表达的影响，以及KCa 3.1在EGF诱导HUVECs体外增殖过程中的作用。 方法 MTT法筛选EGF最佳实验浓度；免疫荧光和Western blotting技术检测EGF对HUVECs细胞KCa 3.1的表达；经KCa 3.1阻断剂TRAM-34处理，MTT法分析EGF诱导的HUVECs增殖情况；流式细胞技术检测细胞周期，RT-PCR法分析细胞周期蛋白D1（cyclinD1）及周期蛋白依赖性蛋白激酶4（CDK4）表达的水平。 结果 EGF处理细胞48h后，MTT实验证实25μg/L浓度的EGF促细胞增殖效应最强。免疫荧光染色显示，EGF明显促进KCa 3.1蛋白表达，且Western blotting提示KCa 3.1的表达上调1.4倍。进一步研究显示，高选择性阻断剂TRAM-34阻断KCa 3.1通道48h后EGF的促细胞增殖作用显著下降，并呈时间和剂量依赖性；细胞周期G1期细胞百分比明显增加；CDK4的mRNA表达下调，而cyclinD1表达无明显变化。结论 KCa 3.1可能通过影响细胞周期进程调节EGF诱导的HUVECs增殖过程。
Objective To examine the effect of epidermal growth factor (EGF) on the expression of intermediate conductance Ca2+ activated K+ channels 3.1 (KCa 3.1) in human umbilical vein endothelial cells (HUVECs), and to investigate the roles of KCa 3.1 in the HUVECs proliferation induced by EGF in vitro. Methods In this present study, the best working concentration of EGF was decided by MTT assay. The expression of KCa 3.1 was tested by immunofluorescence staining and western blotting assay after EGF-treatment. After the HUVECs were treated by TRAM-34, a blocker ofKCa 3.1, the EGF-induced proliferation of HUVECs was determined by MTT assay. Flow cytometry was performed to check the cell cycle of HUVECs. The mRNA expressions of cyclinD1 and cyclin-dependent kinase 4 (CDK4) were investigated by RT-PCR assay. Results After the HUVECs were treated with EGF for 48h, MTT assay showed the maximal stimulation of EGF at 25μg/L on the cell proliferation. EGF increased the expression of KCa 3.1 in immunofluorescence staining, and a significant 1.4-fold increase in KCa3.1-protein levels was revealed by Western blotting. Further research showed，after KCa 3.1 channels were selectively blocked with TRAM-34 for 48hours, the EGF-stimulated proliferation of HUVECs was suppressed observably in a dose-and time-dependent fashion, and the percentage of cells in G1 phase significantly increased in cell cycle. Interestingly, the blocked-KCa 3.1 down-regulated the expression of CDK4 in mRNA levels, whereas the cyclinD1 expression remained unchanged. Conclusions KCa 3.1 could potentially regulate the cell proliferations induced by EGF through modulating the cell cycle process in HUVECs.
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