Effect Of Lentivirus-mediated E1A-activated Gene Repressor(CREG)Gene On Photodamage And Anti-apoptotic Ability Of 661W Cells

Objective:Lentivirus-mediated CREG gene was transfected into 661W cells.The anti-apoptotic ability of 661W cells overexpressing CREG gene modified under light injury was observed.The protective effect of CREG gene on photoreceptor cells was evaluated and its mechanism was discussed.Methods:1.Cultured cells,661W and 293T cell lines were resuscitated,subcultured,frozen and counted.2.Construction of lentivirus-mediated CREG gene expression vector.3.Transfection of 661W cells,Western blot to verify the expression of lentivirus-mediated CREG gene transfection 661W cells;Lentivirus-mediated CREG gene stable transfection of ^CREG661W cells and GFP empty vector control ^GFP661W cells.4.Prepare light damage models of ^CREG661W cells and^GFP661W cells and set normal 661W cells(^Normal661W)as normal blank group.5.Hoechst/PI staining was used to detect the morphological changes of each group,and the morphological differences of each group were observed.BCA method was used to determine the concentration of protein in each group.Western blotting was used to detect the expression levels of different apoptosis-related proteins Caspase3/8/9,Bax and anti-apoptotic protein Bcl-2in each group.6.Pretreatment of each group of cells with P38 inhibitor SB203580,JNK inhibitor SP600125 and ERK inhibitor PD98059.Western blotting was used to detect the expression of key proteins P38,JNK,ERK and their phosphorylated forms in MAPK apoptosis signaling pathway after photodamage.Statistical analysis and quantitative analysis were performed to evaluate whether CREG gene-modified 661W cells inhibit photoreceptor-induced apoptosis and its mechanism of action.Results:1.The pLvx-puro-CREG plasmid was successfully transfected into 293T virus packaging cells and the transfection efficiency was more than 70%.2.Lentivirus was successfully infected to 661W cells,and a stable ^CREG661W cell line was established after repeated screening of purinomycin.3.Detection of CREG protein by Western blot confirmed the successful infection of lentivirus-mediated E1A-activated gene repressor（CREG）gene.4.Cell morphology assay analysis LD^CREG661W compared with other groups LD^Normal661W and LD^GFP661W,apoptosis significantly decreased（P<0.05）.5.Western blot was used to detect the expression level of apoptosis-related proteins in each group.The expression levels of Caspase3/8/9 and Bax in the light injury group were lower than those in the light injury control group（P<0.01）,while the expression of Bcl-2 in the light injury experimental group was significantly higher than that in the light injury control group（P<0.01）.6.Western blot analysis of inhibitors pretreated ^CREG661W cells showed that the ratio of phosphorylation and non-phosphorylated protein expression of JNK,P38 and ERK was lower than that of the control group,the difference between p-JNK/JNK and p-P38/P38 is significant,followed by p-ERK/ERK（P<0.05）.Conclusion:1.^CERG661W cell lines can be stably transfected by lentivirus-mediated CREG gene transfection.2.The ability of retinal photoreceptor-like cells(^CREG661W cells)carrying CREG gene to inhibit apoptosis induced by light injury was significantly enhanced.3.JNK and P38 in the MAPK signaling pathway are the key signaling pathways to inhibit photoreceptor cell apoptosis in retinal photodamage.