人视网膜色素上皮细胞脱分化模型的建立

Establishment of human retinal pigment epithelium（ RPE） cell EMT model

关键词:  上皮-间质转变  视网膜色素上皮细胞  增生性玻璃体视网膜病变

Key words:  epithelial-mesenchymal transition  retinal pigment epithelial  proliferative vitreoretinopathy

摘要:

目的建立人视网膜色素上皮（retinal pigment epithelium,RPE）细胞脱分化的模型,体外模拟增生性玻璃体视网膜病变（proliferative vitreoretinopathy,PVR）中RPE的上皮-间质转化（epithelial-mesenchymal transition,EMT）过程。方法将ARPE-19细胞培养在无血清、含N2和NEAA的petri dish中,建立RPE的EMT模型,采用倒置荧光显微镜观察细胞形态的转变,采用基因芯片和定量PCR来检测EMT相关基因的表达,采用Western blotting和ELISA来检测相关蛋白的表达。结果 ARPE-19细胞在建模后形态明显发生了改变,基因芯片和Q-PCR检测显示一些与EMT相关的因子（如TGF-β1,Vimentin,CD46）及炎症因子（IL-15）和细胞因子（EGF）的表达上调,ELISA在蛋白水平上显示建模后细胞表达的TGF-β1,IL-15,和EGF蛋白明显增多,Western blotting显示Vimentin蛋白的表达也显著上调。结论 ARPE-19在N2和NEAA的petri dish中培养可以诱导EMT的发生。

Abstract:

Objective To establish an epithelial mesenchymal transition（ EMT） model of human retinal pigment epithelium（ RPE） cells. Methods Human retinal pigment epithelium ARPE-19 cells were treated with N2 and nonessential amino acids（ NEAA） in un-coating petridish. Cell morphology was observed with phase-contrast microscope; the expression TGF-β1,vimentin,CD46 genes was measured by gene chip and quantitative PCR; the expression levels of TGF-β1,vimentin,CD46 proteins and inflammatory factors IL-1,IL-15,EGF were detected by Western blotting and ELISA.Results After treatment of N2 and NEAA,the morphology of ARPE-19 cells changed significantly.Gene Chip and Q-PCR confirmed that the expression of EM T related genes TGFβ-1,vimentin,CD46 and inflammatory factors IL-1,IL-15,and EGF were significantly increased. Western blotting analysis confirmed the increase expression of vimentin protein. ELISA showed that the protein levels of TGF-β1,IL-15,and EGF were also significantly increased. Conclusion Treatment with N2 and NEAA in un-coating petridish can induce EMT of ARPE-19 cells.