人视网膜色素上皮细胞增生中表皮生长因子-表皮生长因子受体-有丝分裂原激活蛋白激酶信号传导途径的激活及其作用

Activation and role of signal transduction pathway of epidermal growth factor-epidermal growth factor receptor-mitogen activated protein kinase in proliferation of human retinal pigment epithelial cells

目的 观察人视网膜色素上皮 (RPE)细胞增生中表皮生长因子 (EGF) -表皮生长因子受体(EGFR) -有丝分裂原激活蛋白激酶 (MAPK)信号传导途径的激活及其作用。 方法 人 RPE细胞分别在0 .1%、10 %小牛血清 (FCS) ,EGF(0 .1、1、10、5 0、10 0 ng/ ml)及上述各浓度 EGF与 10 %FCS联合作用下培养 3d。采用原位杂交及免疫细胞化学方法 ,检测 RPE细胞 EGFR m RNA及蛋白质表达。使用抗磷酸化细胞外信号调节激酶 (ERK) 1/ 2抗体进行免疫细胞化学染色 ,以观察 MAPK激活情况。 结果 EGF在10 %FCS改良 Eagle培养液 (DMEM)中作用时 ,其最佳刺激浓度为 1ng/ ml,EGF在 0 .1%FCS DMEM中作用时最佳作用浓度为 10 ng/ ml;EGF作用后 ,磷酸化 ERK 1/ 2抗体由在细胞浆中表达转移至在细胞核中表达。 结论 EGF呈浓度依赖性地促进 RPE细胞 EGFR m RNA及蛋白质的表达 ,且对 MAPK也具有浓度依赖性的核转位作用。推测 EGF- EGFR- MAPK信号传导途径可能在 RPE细胞增生中起重要作用 ,血清在此过程中有明显的促进作用。

Objective To investigate the activation and role of signal transduction pathway of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR)-mitogen activated protein kinase (MAPK) in proliferation of human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were stimulated with 0.1%,10% foetal calfserum (FCS) and EGF(0.1, 1, 10, 50 and 100 ng/ml)in 0.1% FCS Dulbeco′s modified Eagle′s medium (DMEM) and in 10% FCS DMEM for 3 days, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and EGFR mRNA,respectively. Activation of MAPK was detected by immunohistochemical method with specific anti-phosphorylated ERK 1/2 antibody. Results The optimal concentrations of EGF were 10 ng/ml in 0.1% FCS DMEM and 1 ng/ml in 10% FCS DMEM. After 3 days of stimulation with EGF, phosphorylated ERK 1/2 staining was detectable in nucleus of RPE cells, whereas cells presented immunostaining for phosphorylated ERK 1/2 in the cytoplasm before stimulation. Conclusions EGF may improve the expression of EGFR protein and EGFR mRNA of RPE cells, and induced MAPK nuclear translocation in a concentration-dependent manner. EGF-EGFR-MAPK signal transduction pathway may play a key role in RPE cells proliferation, and serum exerts an important acceclerating function in the process.