Study On Apoptosis Of Target CaMK? Gene After Rat Liver Transplantation

Objective:This study was divided into two parts.The first part explored the effect of CaMK? expression on the apoptosis of rat hepatocyte BRL-3A.The second part explored the effect of CaMK? expression on liver function and apoptosis after liver transplantation in rats.Methods:(1)By cultured rat hepatocytes BRL-3A cells stably passage,to construct CaMK??shRNA lentiviral(group A)and CaMKIIy protein(group B)expression system,then transferred then into rat hepatocytes BRL-3A and the corresponding blank vector(C,D group)and normal saline(group E)were perfused into the control group at the same time.The expressions of CaMK?,Cyt C and AIF proteins were detected by Western blot method,and the apoptosis rate of rat hepatocytes BRL-3A was measured by Tunel method.(2)Allogeneic orthotopic liver transplantation model was established by using the classic two-cuff method.The lentiviral expression systems of CaMK??yshRNA(group A)and CaMKIIy protein(group B)were constructed.The lentiviral vector expressing CaMKIIy shRNA and the lentiviral vector expressing CaMK?? protein were perfused into the rat after liver transplantation respectively,and the corresponding blank vector(C,D group)and normal saline(group E)were perfused into the control group at the same time.The serum levels of ALT and AST were measured at different time points of inferior vena cava blood in rats.The expression of CaMK?,Cyt C,AIF protein and hepatocyte apoptosis rate were measured by rat liver.Results:(1)In vitro experiments the protein expression of CaMKII,Cyt C and AIF in group A was significantly lower than that in group E(p<0.05).The protein expression of CaMKII,Cyt C and AIF in B group was significantly higher than that in group E(P<0.05).There was no significant difference between group C,group D and group E(p>0.05).At the same time the apoptosis of liver cell in group A was less of liver cell apoptosis was less than the E group,the difference was statistically sithan than the group E,the difference was statistically significant(p<0.05).At the same time,the apoptosis of hepatocytes in group B was more than that in group E,the difference was statistically significant(p<0.05).There was no significant difference between group C,group D and group E(p>0.05).(2)The levels of serum ALT and AST in the lentiviral vector group(group A)were significantly lower than those in the perfused saline group(group E)after transplantation in the cecal vein of rats after liver transplantation,and the rats were injected with CaMKIIy shRNA(P<0.05).The expression of CaMK?,Cyt C and AIF in group A was significantly lower than group E(P<0.05)and the apoptosis rate of hepatocytes was lower than group E too.The serum levels of ALT and AST in the CaMK?? lentiviral group(group B)were higher than group E(P<0.05),the expression of CaMK?,Cyt C and AIF in group B were significantly higher than group E(P<0.05)and the apoptosis rate of hepatocytes was higher than group E.There was no significant difference in serum ALT and AST levels,CaMK?,Cyt C,AIF protein expression and hepatocyte apoptosis rate after transplantation between C,D,E group(p>0.05).Conclusions:(1)In vitro experiments,blocking CaMK? signaling pathway can inhibit liver cell BRL-3A apoptosis;and enhanced CaMK ? signaling pathway is to promote liver cell BRL-3A apoptosis.(2)Specific blocking CaMK? signaling pathway can effectively reduce the levels of serum ALT,AST and the protein expression of Cyt C,AIF,and the apoptosis rate in liver cells;and enhanced CaMK ?signaling pathway is to increase the levels of serum ALT and AST and the expression of Cyt C and AIF protein in hepatocytes after liver transplantation and the rate of apoptosis.In vitro and in vivo experiments have shown that inhibition of CaMK ?expression can reduce the liver function after liver transplantation and liver cell apoptosis,for the clinical treatment of liver transplantation after liver dysfunction and liver cell apoptosis prevention and treatment to provide a new theoretical basis.