表皮生长因子诱导人视网膜色素上皮细胞整合素α5表达对细胞增生和迁移的影响

摘要

Objective To observe the effect of epidermal growth factor (EGF) on integrin α5 expression and its influence on human retinal pigment epithelium (RPE) cells. Methods Human RPE cells were treated in vitro with 0.1, 1.0, 10.0, 20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin α5 was measured by reverse transcription polymerase chain reaction(RT-PCR)and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12, DMEM/F12 + 10 ng/ml EGF, DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human integrin α5 antibody (1: 100), DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human vimentin antibody (1: 100), and their proliferation and migration were measured by methyl-thiazole tetrazolium(MTT)and Boyden chamber. Results The integrin α5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P = 0. 687), however it was induced in a dose-dependent manner after 24 and 48 hours of EGF stimulation (F=465. 303, 212. 340; P= 0. 000, 0. 000). The protein level of integrinα5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0. 1 ng/ml group (P<0. 01). MTT and Boyden chamber showed that the integrin α5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin α5 expression, thus increase the proliferation and migration of human RPE cells.%目的 观察表皮生长因子(EGF)诱导人视网膜色素上皮(RPE)细胞整合素α5表达对细胞增生和迁移的影响.方法 在0.1、1.0、10.0、20.0、100.0 ng/ml EGF浓度条件下,体外培养人RPE细胞.逆转录聚合酶链反应(RT-PCR)及流式细胞术观察不同浓度组整合素α5mRNA和蛋白的表达.后将实验分为Dulbecco改良的Eagle培养基(DMEM)/F12、DMEM/F12+10.0 ng/ml EGF、DMEM/F12+10.0ng/ml EGF+兔抗人整合素α5多克隆抗体(1:100)及DMEM/F12+10.0 ng/ml EGF+兔抗人波形蛋白抗体(1:100)组,采用噻唑蓝(MTT)比色法和Boyden小室法检测细胞增生及迁移.结果 RT-PCR检测显示,细胞培养12 h,EGF对人RPE细胞整合素α5mRNA合成无明显影响(F-0.618,p=0.687);细胞培养24、48 h,EGF能刺激人RPE细胞合成整合素α5mRNA,并呈浓度依赖性(F=465.303,212.340;P=0.000,0.000).流式细胞术检测显示,10.0 ng/ml组整合素α5荧光强度最强,与空白对照组和0.1 ng/ml组比较,差异有统计学意义(P<0.01).MTT比色法和Boyden小室法检测显示,整合素α5的表达促进人RPE细胞增生和迁移.结论 EGF促进整合素α5mRNA和蛋白的表达,整合素α5的表达促进人RPE细胞增生和迁移.