Effects Of HGF Expression In Human Retinal Pigment Epithelial Cells On Proliferation,Migration And Angiogenesis Of HUVEC

Part 1Objective:To investigate the expression of HGF and VEGF in RPE cells at different time intervals under hypoxia and the effects of exogenous HGF on proliferation,migration and angiogenesis of human umbilical vein endothelial cells(HUVEC)in vitro.Methods:Human RPE cells were cultured in vitro,and eighth to 12th generations of cells with good growth were selected.300?M CoC12 was added at 3h,6h,12h and 24h to induce chemical hypoxia in RPE cells.After 12h the mRNA level of HGF and VEGF were detected by Real-time PCR method,and the protein expression level of HGF and VEGF were detected by Western.The HUVEC cells were randomly divided into normal and control group,10ng/ml HGF protein treatment group and 25ng/ml HGF protein treatment group,using MTT method,scratch test and transwell migration test and in vitro angiogenesis assay to detect the effects of different concentrations of HGF on HUVEC proliferation,migration and tube formation ability.Results:Real-time PCR showed that,compared with the normal control group,hypoxia treated RPE cells after 12h VEGF and HGF levels reached the highest value,and there is a significant difference compare to baseline(P<0.05,P<0.01);Western blot showed that the 6h expression of VEGF after hypoxia treatment the highest 12h protein expression of HGF was the highest.After treatment with lOng/ml HGF and 25ng/ml HGF,the cell proliferation,lumen formation ability and cell migration ability of HUVEC were significantly higher than those of the control group,but there was no significant difference between the HGF treated groups.Conclusion:Hypoxia can induce the expression of HGF and VEGF in RPE cells,and exogenous HGF can promote the proliferation,migration and angiogenesis of HUVEC cells.Part 2Objective:To investigate the effect of HGF expression on proliferation,migration and angiogenesis of RPE cells in co cultured HUVEC cells.Methods:Human RPE cells were cultured in vitro,and eighth to 12th generations of cells with good growth were used for the experiment.With the same generation of RPE cells were randomly divided into normal control group,HGF siRNA treatment group and siRNA control group,the treated RPE cells and normal HUVEC cells were cultured in transwell cell and the upper chamber,to continue training for 12h,24h,48h at the temperature of 37 incubator.VEGF and HGF gene of RPE cells was measured by Real-time PCR method in the level of mRNA.MTT,scratch test,transwell migration assay and in vitro vasculogenesis test were used to detect the migration,tube formation ability and proliferation of HUVEC cells.Results:Compared with normal control group,VEGF and HGF gene expression of siRNA negative control group did not change significantly;compared with the siRNA negative control group,the expression of VEGF and HGF gene in HGF siRNA group were decreased by 57%,70%.MTT results showed that the HUVEC cell activity of siRNA negative control group did not change significantly,HGF siRNA treatment group significantly decreased cell viability of 24h and 48h after co-culture;scratch test and Transwell migration test results show that the cell migration ability of HGF siRNA treatment group was significantly inhibited,in vitro angiogenesis assay results showed that HGF siRNA treatment inhibited the in vitro angiogenesis ability of HUVEC significantly.Conclusion:HGF can promote the proliferation,migration and angiogenesis of HUVEC cells,and the mechanism of its effect on ocular neovascular diseases needs further study.