Study On The Antioxidation Of Human ApoE3 Gene Transfected To PC12 Cells

Objectives:In this study,we constructed pEGFP-N1-H-ApoE3 neural cell model in vitro,simulated oxidative stress injury by H2O2;detected cell survival rate by CCK-8,and detected cell apoptosis by Hochest33258 and flow cytometry,and explored the repair of ApoE3 and its possible mechanism of action.Then we will clarify the important role of ApoE3 in secondary spinal cord injury.Methods:In this study,rat pheochromocytoma cells(PC 12 cells)were selected as neural cell models and cultured in 10%DMEM high glucose medium.The experiment was divided into four groups:(1)normal control group(normal nerve cell culture);(2)H2O2 group(normal nerve cell culture + H2O2injury);(3)H2O2+ empty plasmid group(transfected pEGFP-N 1+H2O2 injury);H2O2+ApoE3 group(transfected pEGFP-N1-H-ApoE3+ H2O2 injury).The stable expression of ApoE3 positive clones was screened by G418 screening,and the stable expression of ApoE3 protein was determined by Western blot analysis(Western-Blot analysis).The positive clone was amplified and cultured,and the cell survival rate was detected by CCK-8 cell proliferation and toxicity test kit.The apoptosis rate of cells was detected by Hochest33258 reagent and flow cytometr。Results:1.After 2 weeks of continuous screening with G418,the positive clone appeared.The cells could be passaged steadily.Under fluorescence microscope,the distribution of green fluorescence was homogeneous.Western blot showed that ApoE3 protein expression increased significantly.2.CCK-8 cell proliferation toxicity test kit was used to detect the cell viability of H2O2,H2O2 + empty plasmid and H2O2 +ApoE3 group(P<0.05)compared with the normal control group.Compared with group H2O2,cell survival rate of H2O2 + empty plasmid group was not significantly different(P>0.05).Compared with group H2O2 and H2O2 + empty plasmid group,cell survival rate of H2O2 +ApoE3 group increased significantly(P<0.01).3.Hochest33258 staining showed that the normal PC12 cell circle of pale blue,intact nucleus,nucleus of apoptotic cells bright white fluorescence,karyopyknosis,and even broken;the normal control group apoptosis rarely,other cell apoptosis was obvious,but apoptotic cells in H2O2 +ApoE3 group were significantly lower than that in H202 group and H2O2+ plasmid group decreased.4.Detection of cell apoptosis rate by flow cytometry was found:Compared with the normal control group,the apoptosis rate of H2O2 group,H2O2+ empty plasmid group and H2O2+ApoE3 group increased(P<0.01).Compared with H2O2group,there was no significant difference in apoptosis between H2O2+ empty plasmids group(P>0.05).Compared with H2O2group and H2O2+ empty plasmid group,the early apoptosis rate of H2O2+ApoE3 group decreased(P<0.05),The apoptosis rate of H2O2+ApoE3 group was not significantly different from that of the other two groups(P>0.05).Conclusion(s):(1)It is found that ApoE3 protein can improve the survival rate of PC12 cells after oxidative stress.It is suggested that ApoE3 protein has certain antioxidant damage ability.(2)We found that ApoE3 protein can effectively reduce the early apoptosis rate of PC12 cells,but it has no significant protective effect on apoptosis in advanced stage.It is suggested that ApoE3 protein plays a positive protective role in the repair of nerve injury.