The Protective Effect And Mechanism Of HO-1 Gene Transfection On Human Retinal Pigment Epithelium Injury Caused By Apoptosis In Vitro

Objective1.Construction of Lentivirus vector（LV）-mediated ARPE-19 heme oxygenase-1（HO-1）recombinant overexpression virus（LV-HO-1）and transfection of it into ARPE-19 cells;2.To investigate the protective effect of LV-HO-1 on RPE cell apoptosis.3.To explore the protective mechanism of LV-HO-1 on RPE cell apoptosis.Method1.Construction of recombinant lentiviral vector overexpressing HO-1;2.Establish the apoptotic model of RPE:24 hours as a single apoptotic time,and the half inhibitory concentration of H₂O₂（IC50=209umol H₂O₂）as the optimal apoptotic concentration in normal cultured RPE cells.Establish the apoptotic model of H₂O₂3.RPE cells were divided into four groups according to different treatment factors:Blank control group:normal cultured RPE cells;pure injury group:normal RPE cells apoptosis induced by hydrogen peroxide;empty vector-RPE group:lentivirus-mediated empty vector transfection of RPE cells;HO-1-RPE group:lentivirus-mediated HO-1gene transfection of RPE cells;4.Cell counting method was used to plot the growth curve of RPE cells in each group.5.Fluorescence microscopy was used to observe the expression of fluorescence signal in RPE cells of each group;CCK-8 reagent was used to detect the survival rate of RPE cells after injury;Flow cytometry and mitochondrial membrane potential（MTP）were used to detect the apoptosis of RPE cells in each group;transmission electron microscopy was used to detect the apoptotic ultrastructure of RPE cells in each group.Western blot was used to detect the expression of Nrf2,HO-1,Bcl-2 and Caspase-3.Result1.The recombinant lentivirus vector overexpressing HO-1 was successfully constructed and transfected into RPE cells.2.The apoptotic model of H2O2 was successfully established.T3.Survival rate（SR）of HO-1-RPE group was significantly higher than that of pure injury group and empty carrier-RPE group（P<0.01）.4.here was no significant difference in growth curve between HO-1-RPE group and pure injury group and empty carrier-RPE group（P>0.05）.5.Apoptosis rate（AR）in HO-1-RPE group was significantly lower than that in pure injury group and empty carrier-RPE group（P<0.01）.6.Apoptosis rate（AR）of HO-1-RPE group was significantly lower than that of pure injury group and empty carrier-RPE group（P<0.01）.7.The ultrastructure of apoptotic cells in HO-1-RPE group was significantly improved compared with that in pure injury group and empty carrier-RPE group.8.The expression of apoptotic pathway-related proteins Nrf2,HO-1 and Bcl-2 in HO-1-RPE group was significantly higher than that in pure injury group and empty carrier-RPE group,while Caspase 3 was significantly lower（P<0.01）.Conclusion1.The HO-1 gene was successfully transfected into RPE by lentivirus packaging system2.HO-1 gene transfection and protein expression can improve the survival rate of RPE under the apoptotic model of H₂O₂,and have protective effect against apoptotic cells induced by apoptotic injury in vitro.3.HO-1 can resist apoptosis induced by H₂O₂ injury,and confirm that the prote ctive mechanism can be achieved by regulating the nuclear factor Nrf2/HO-1/bcl-2/Caspase-3 pathway.