摘要
目的探讨表皮生长因子(EGF)对人视网膜色素上皮细胞(hRPE)表达整合素α5的调节及意义。
方法1)通过玻璃体切除术采集14例视网膜脱离病例的视网膜下膜标本,用免疫组化方法检测下膜中角蛋白,整合素α5β1及纤维粘连蛋白(FN)的表达。免疫荧光双标记法研究视网膜下膜色素上皮细胞与整合素α5β1表达的关系,荧光定量分析视网膜下膜与正常视网膜下膜组织中整合素α5β1表达的差异。2)人视网膜色素上皮细胞原代、传代培养;分别用0ng/ml、0.1 ng/ml、1 ng/ml、10 ng/ml、20 ng/ml、100 ng/ml浓度EGF诱导hRPE;RT-PCR、免疫组化和流式细胞术观察整合素α5mRNA和蛋白的表达;MTT法检测各实验组细胞增生率,采用直径为8.5mm的Boyden小室实验进行细胞迁移能力的检测。3)将实验分成对照组、10ng/mlEGF组和PD98059组,RT-PCR及流式细胞术观察不同实验组整合素α5 mRNA和蛋白的表达;Western blot法检测各组hRPE细胞中MAPK磷酸化水平。
结果1)所有14例下膜标本中整合素α5β1纤维粘连蛋白(FN)表达均呈阳性,角蛋白表达均呈阳性的细胞(即RPE细胞)的细胞膜上表达整合素α5β1,与正常视网膜组织比较表达显著增强(P<0.05)。2)与0ng/ml组相比,24h后1ng/ml的EGF促进人hRPE细胞中整合素α5mRNA和蛋白的表达,并呈浓度依赖性,而浓度为10～100ng/ml时促进作用达到高峰。流式细胞术检测显示:10ng/ml的EGF作用时荧光强度最强为3.98±0.67,0ng/ml和0.1ng/ml组分别为1.87±0.22,1.98±0.54(P<0.01)。MTT法检测显示:与对照组相比,EGF组及加入不相关抗体(兔抗人波形蛋白抗体)组细胞增生、迁移明显,而加入兔抗人整合素α5多克隆抗体(1:100)组较弱,与前两组相比差异有显著性(P<0.01)。3)与对照组相比,EGF促进整合素α5 mRNA和蛋白的表达,而PD98059(20μmol/L)可抑制此促进作用;流式细胞术显示24 h后整合素α5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结果显示30 min后EGF组ERK1/2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。
结论在视网膜下膜组织中hRPE细胞整合素α5β1表达上调,其配体FN也显著表达,整合素α5β1与FN参与了视网膜下膜的形成。1ng/ml的EGF开始促进整合素α5mRNA和蛋白的表达,10～100ng/ml时候促进作用达到高峰,整合素α5的表达促进hRPE细胞的增生和迁移。ERK1/2的磷酸化激活参与此过程。EGF激活ERK1/2通路刺激hRPE细胞的整合素α5mRNA的表达是PVR的重要发病机制。
Objective To investigate the significance and regulation of epidermal growth factor (EGF) on human retinal pigment epithelial cells’(hRPE) integrinα5 expression.
Method 1) We collected 14 subretinal membrane specimens by vitrectomy of retinal detachment cases.And detected the expressions of keratin, integrinα5β1 and fibronectin (FN) by immunohistochemical methods. We studied the relation between retinal pigment epithelial cell membranes and expression of integrinα5β1 by double-labeling immunofluorescence,and analyzed the difference of integrinα5β1 expression of retinal membrane with the membrane compared with normal by fluorescence quantitative. 2)We used human retinal pigment epithelial cells of primary and mass culture.The hRPE cells were induced by EGF of different concentrations (0ng/ml,0.1ng/ml, 1ng/ml, 10ng/ml, 20ng/ml,100ng/ml).We observed the integrinα5 mRNA and protein expression by RT - PCR, immunohistochemistry and flow cytometry .We measured cells proliferation rates of the experimental groups by MTT assay,and tested cells migration with a diameter of 8.5mm Boyden chamber. 3) There were three groups: the control group, the 10ng/mlEGF group and the PD98059 group. We observed the integrinα5 mRNA and protein expression of different groups by RT-PCR and flow cytometry, and tested hRPE cells MAPK phosphorylation level in each group by Western blot.
Result 1) All 14 cases were positive expression of integrinα5β1 and fibronectin (FN) protein,the RPE cells with keratin expressedα5β1 on the subretinal membrane, compared with the normal the expression significantly enhanced(P <0.01). 2)Compared with 0ng/ml group, the 1ng/ml of EGF promoted integrinα5 mRNA and protein expression in hRPE cells after 24 hours and with a concentration-dependent manner;and the role peaked at concentrations of 10 to 100ng/ml. Flow cytometry showed that the 10ng/ml of EGF played the strongest role in integrinα5 induction;and the fluorescence intensity was 3.98±0.67.When the concentrations were 0ng/ml and 0.1ng/ml,they were1.87±0.22, 1.98±0.54 respectively (P <0.01). MTT assay showed results as follows: compared with the control group,cells proliferated and migrated obviously in EGF-induced group and not-related antibody (rabbit anti-human antibody vimentin) one;the role was weak in rabbit anti-human integrinα5 polyclonal antibody (1:100) group; and the difference was significant (P <0.01). 3) Compared with the control group, EGF could promote expression of integrinα5 mRNA and protein, but PD98059 (20μmol/L) inhibited this role. Flow cytometry showed the integrinα5 fluorescence intensity after 24 hours were 1.94±0.22, 4.56±0.25, 2.39±0.14, and the difference was significant (P <0.05). After 30 minutes: Western blot results showed that the highest phosphate levels of ERK1/2activation in EGF-induced group, and the control group of the ERK1/2 phosphorylation activated weak, but the PD98059 inhibited the activation.
Conclusion The hRPE cells in the subretinal membranes up-regulated expression of integrinα5β1, and its ligand FN also notably expressed ; and so the integrinα5β1 and FN played the role in the retinal membrane formation. 1ng/ml of EGF promoted integrationα5 mRNA and protein expression and the promotion reached a peak of 10~100 ng/ml; and the expression of integrinα5 in hRPE promoted cells proliferation and migration. ERK1/2 phosphorylation participated in the activation process. EGF stimulated activation of ERK1/2 pathway in hRPE cells, and it promted the integrinα5 mRNA expression in playing an important role in PVR.
引文
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