The Role Of P53 SIRNA Transfection In Apoptotis And Ubiquitination Of Glomerular Podocyte With High Glucose Induced

Objective: This study cultured the mice podocyte in vitro,transfected p53 siRNA and observed the influence of podocyte apoptosis and ubiquitination in high levels of glucose.In order to make clear the effect of p53 and COP1-mediated ubiquitin proteasome pathway in podocyte apoptosis induced by high levels of glucose, clarify the pathogenesis of diabetic nephropathy.Methods: Cultured the mice podocytes in vitro. Divided into control group, high sugar group, mannitol group, p53 siRNA plasmid group, p53 siRNA plasmid negative group. TUNEL method detect the change of podocyte apoptosis, RT - PCR and western blot detect the expressions of p53, COP1 and caspase-3 mRNA and protein.Results:(1) The apoptosis index of podocytes in high glucose group was (14.4±1.67)% ,which was 4.8 and 2.6 folds respectively higher in normal group(3.003±1.00)% and PFT-αgroup(5.60±1.67)%, mannital group(14.8±2.28)% and p53 siRNA plasmid negative group(15.6±2.97) % were the same as high glucose group.(2) After 48 hours high glucose stimulating, p53 mRNA (1.2351±0.1005) was higher than the control group(0.5085±0.0059) (P < 0.05), COP1 mRNA (0.7974±0.0192) was lower than the control group (1.3336±0.0903) (P < 0.05), caspase-3 mRNA (0.4376±0.0625) was higher than the control group(0.1152±0.0485) (P < 0.05). After transfecting p53 siRNA we found p53 mRNA (0.5283±0.0099) and caspase-3 mRNA(0.1866±0.0981)were lower than high glucose group (P < 0.05), COP1 mRNA (0.83±0.028) was the same as high glucose group (P > 0.05). The expression of p53, COP1 and caspase-3 mRNA in mannitol group, p53 siRNA plasmid negative group and high glucose had no difference(P>0.05).(3) After 48 hours high glucose stimulating, p53 protein (0.6921±0.0944) was higher than the control group(0.2420±0.0392) (P < 0.05), COP1 protein (0.135±0.03848) was lower than the control group (0.3359±0.0865) (P < 0.05), caspase-3 protein (0.576±0.1014) was higher than the control group(0.226±0.0131) (P < 0.05). After transfecting p53 siRNA we found p53 protien (0.1002±0.0349) and caspase-3 protein(0.138±0.011)were lower than high glucose group (P < 0.05), COP1 protein (0.142±0.03518) was the same as high glucose group (P > 0.05). The expression of p53,COP1 and caspase-3 protein in mannitol group, p53 siRNA plasmid negative group and high glucose had no difference(P>0.05).Conclusion:(1) High levels of glucose could induce apoptosis in mouse podocyte.The mechanism maybe that high levels of glucose increase the expression of p53 and inhibite COP1-mediated ubiquitination pathway in mouse podocyte.(2) Reduced the level of p53 gene could decrease podocyte apoptosis, but the COP1-mediated ubiquitin proteasome pathway unchanged.