目的:探讨厄洛替尼抑制表皮生长因子受体(EGFR)和EGFRsiRNA对表皮生长因子(EGF)诱导的视网膜色素上皮(RPE)细胞活化的影响。
方法:人RPE细胞系(ARPE-19细胞)被100ng/mL的EGF活化。厄洛替尼和EGFRsiRNA介入EGF医治。细胞活力,扩散,并通过甲基噻唑基四唑(MTT)测定法检测迁移,溴脱氧尿苷(BrdU)染色试验和伤口愈合试验,分别。EGFR/蛋白激酶B(AKT)通路蛋白和N-钙黏着蛋白,α-平滑肌肌动蛋白(α-SMA),和波形蛋白用Westernblot检测。EGFR也通过免疫荧光染色确定。
结果:EGF处理24h诱导ARPE-19细胞活力显著增加,扩散和迁移,EGFR/AKT蛋白磷酸化,并降低总EGFR表达。厄洛替尼抑制ARPE-19细胞的活力,通过下调EGFR和AKT总蛋白表达来实现增殖和迁移。厄洛替尼还抑制EGF诱导的ARPE-19细胞增殖和迁移能力的增加,并明显抑制EGF诱导的EGFR/AKT蛋白磷酸化和N-cadherin的表达降低,α-SMA,和波形蛋白。同样,EGFRsiRNA抑制EGFR显著影响EGF诱导的细胞增殖增加,生存能力,和移民,EGFR/AKT蛋白磷酸化,和N-钙粘蛋白的上调,α-SMA,和波形蛋白。
结论:厄洛替尼和EGFR敲低抑制EGF诱导的细胞活力,扩散,和通过EGFR/AKT途径在RPE细胞中的迁移。EGFR抑制可能是增殖性玻璃体视网膜病变(PVR)的一种可能的治疗方法。
OBJECTIVE: To explore the effect of epidermal growth factor receptor (EGFR) inhibition by erlotinib and EGFR siRNA on epidermal growth factor (EGF)-induced activation of retinal pigment epithelium (RPE) cells.
METHODS: Human RPE cell line (ARPE-19 cells) was activated by 100 ng/mL EGF. Erlotinib and EGFR siRNA were used to intervene EGF treatment. Cellular viability, proliferation, and migration were detected by methyl thiazolyl tetrazolium (MTT) assay, bromodeoxyuridine (BrdU) staining assay and wound healing assay, respectively. EGFR/protein kinase B (AKT) pathway proteins and N-cadherin, α-smooth muscle actin (α-SMA), and vimentin were tested by Western blot assay. EGFR was also determined by immunofluorescence staining.
RESULTS: EGF treatment for 24h induced a significant increase of ARPE-19 cells\' viability, proliferation and migration, phosphorylation of EGFR/AKT proteins, and decreased total EGFR expression. Erlotinib suppressed ARPE-19 cells\' viability, proliferation and migration through down regulating total EGFR and AKT protein expressions. Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin, α-SMA, and vimentin proteins. Similarly, EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation, viability, and migration, phosphorylation of EGFR/AKT proteins, and up-regulation of N-cadherin, α-SMA, and vimentin proteins.
CONCLUSIONS: Erlotinib and EGFR-knockdown suppress EGF-induced cell viability, proliferation, and migration via EGFR/AKT pathway in RPE cells. EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy (PVR).