The Culture And Characterization Of Primary Human RPE Cells And The Mechanism Of YM155 On Human RPE Cells

Objective: In this study,we isolated and cultured primary human RPE cells and characterized,and compared the growth properties of primary human RPE cells and ARPE19 cells.We investigated the effects and mechanism of YM155 on cell survival,proliferation,and migration of primary human RPE cells and ARPE19 cel s.Methods: Primary human RPE cells were isolated and cultured by trypsin digestion method.The growth curve of primary human RPE cells and ARPE19 cells was tested by i C ELLigence real-time cell analyzer(RTC A).The protein of RPE65,CRALBP,ZO-1 and EGFR in primary human RPE cells and ARPE19 cells was detected by immunofluorescence staining.The expression of EGFR in primary human RPE cells and ARPE19 cells was interfered with si RN A.The effect of YM155 on the primary human RPE cells and ARPE19cells' viability was detected by MTT and flow cytometry assay.The effect of YM155 on the primary human RPE cells and ARPE19 cells' proliferative capacity was tested with Brdu tagged incorporation assay.The effect of YM155 on the primary human RPE cells and ARPE19 cells' migrative ability was detected by wound-healing assay.The effect of YM155 on the expression of EGFR in primary human RPE cells and ARPE19 cells was examined by immunofluorescence assay.The effect of YM155 on the protein of EGFR?ERK?p-ERK?JNK?p-JNK?P38?p-P38 was tested by immunoblotting assay.Results: The primary human RPE cells were successfully isolated and cultured by trypsin digestion method and the harvested primary human RPE cells showed a high purity.Immunofluorescence staining assay showed that RPE65,CRALBP,ZO-1 and EGFR proteins were significantly expressed in primary human RPE cells and ARPE19 cells.The cell index of ARPE19 cells was higher than that of P3 and P4 primary human RPE cells.The si RNA interference assay showed that EGFR si RNA had a stronger down-regulation effect on EGFR protein in primary human RPE cells than that in ARPE19 cells.MTT showed that YM155 suppressed primary human RPE cells and ARPE-19 cells' viability in a time and concentration-dependent manner.Flow cytometry assay showed that a low dose of YM155 had no significant effect on the death of primary RPE and ARPE19 cells,a high dose of YM155 induced a small amount of ARPE-19 cell apoptosis and necrosis.Brdu tagged incorporation assay showed that YM155 inhibited the proliferation of primary human RPE cells and ARPE19 cells and also inhibited EGF-induced promotion of the proliferation in primary human RPE cells and ARPE19 cells.Wound-healing assay shows that YM155 inhibited the migration of primary human RPE cells and ARPE19 cells and also inhibited EGF-induced promotion of migration of primary human RPE cells and ARPE19 cells.Immunoblotting assay showed that YM155 down-regulated total EGFR and phosphorylated ERK in primary human RPE cells and ARPE19 cells,and up-regulated the phosphorylation of P38 MAPK and phosphorylated JNK.In addition,YM155 inhibited EGF-induced expression increase of phosphorylated EGFR protein and phosphorylated ERK protein.Immunofluorescence staining assay showed that YM155 induced EGFR endocytosis in primary human RPE cel s and ARPE19 cel s.Conclusions: High purity primary human RPE cells can be successfully cultured by trypsin digestion method.RPE65,CRALBP,ZO-1 and EGFR protein were expressed in primary human RPE cells and ARPE19 cells.The ARPE19 cells' proliferation ability was stronger than primary human RPE cells.The inhibitory effect of YM155 on the survival,proliferation and migration of primary human RPE and ARPE-19 cells is closely related to the EGF/EGFR/MAPK signaling pathway,YM155 might be a potential inhibitor for the abnormal survival of RPE cells in PVR.