EGF诱导人RPE细胞表达整合素α_5过程中丝裂原激活的蛋白激酶的作用

The effects of mitogen-activated protein kinase on EGF-induced expression of integrin α_5 in human RPE cells

目的探讨表皮生长因子(EGF)诱导人视网膜色素上皮(RPE)细胞表达整合素α5过程中丝裂原激活的蛋白激酶(MAPK)的作用。方法将人RPE细胞分为对照组、EGF组和PD98059组,RT-PCR及流式细胞术观察各组整合素α5mRNA和蛋白的表达;Western blot法检测各组人RPE细胞中MAPK的磷酸化水平。结果对照组的整合素α5mRNA/β-actin mRNA像素值的比值为0.93±0.06,PD98059组为1.06±0.07,EGF组为1.97±0.09,EGF组与其他2组相比,差异有统计学意义(P<0.01);流式细胞术检测显示,24 h后整合素α5的荧光强度分别为1.94±0.22、4.56±0.25、2.39±0.14,差异有统计学意义(P<0.05)。Western blot检测显示,30 min后EGF组细胞外信号调节激酶(ERK)1/2的磷酸化激活水平最高,PD98059组抑制ERK1/2的磷酸化激活,对照组ERK1/2的磷酸化激活作用很弱。结论ERK1/2的磷酸化激活参与EGF诱导人RPE表达整合素α5的过程。EGF激活ERK1/2通路刺激人RPE细胞整合素α5mRNA的表达。

Background Human retinal pigment epithelial （RPE） cells play a critical role in the pathogenesis of proliferative vitreoretinopathy （PVR） and other related ocular diseases. Research demonstrated that epidermal growth factor（EGF） stimulates activation of RPE cells and therefore causes PVR,and integrin α5 mediates the adhesion of cells and EGF. Objective This study aims to investigate the role of mitogen-activated protein kinase（MAPK） in regulation of EGF on integrin α5 expression in human RPE cells. Methods Human RPE cells strain（CRL2302） was cultured in DMEM/F12 with 10% calf serum and passaged in 0.25% trypsin. Cultured cells were divided into DMEM/F12control group,20 μmol/L PD98059 ＋ 10 ng/mL EGF ＋ DMEM/F12（PD98059） group and 10 ng/mL EGF ＋ DMEM/F12 （EGF） group. The expression of integrin α5 protein in CRL2302 cells was detected by RT-PCR and calculated as α5 mRNA/13-aetin mRNA,and the expression of integrin α5 mRNA in CRL2302 cells was detected evaluated by flow cytometry. The MAPK phosphorylation level in each group of human RPE cells was tested by Western blot. Results The expression value of integrin α5 mRNA was 0.93 ± 0.06 in the control group, 1.06 ± 0.07 in PD98059 group and 1.97 ± 0. 09 in EGF group, showing a significant difference among the three groups （ F = 458. 896, P 〈 0. 01 ）. The fluorescence intensity of integrin α5 protein in CRL2302 cells after 24 hours was 1.94 ± 0.22,4.56 ± 0.25,2.39 ± 0. 14 in three groups respectively with a significant difference （ F = 21.05, P 〈 0.05 ）. After 30 minutes of culture, Western blot result showed that the strongest phosphorylation levels of ERK1/2 activation in EGF group and the weakest phosphorylation levels of ERK1/2 activation in the control group;While that in PD98059 group was significantly stronger than control group and weaker than EGF group（F = 143.14 ,P 〈 0. 01 ）. Conclusion EGF stimulates activation of ERK1/2 pathway in human RPE cells and the expressions of integrinα5 mRNA and protein in human RPE cells in vitro.