A Preliminary Study On The Role Of CaMK? In Apoptosis And Mitochondrial Apoptosis After Rat DCD Liver Transplantation

Part?A summary of donation after cardiac death orthotopic liver transplantation model in ratsObjective(s):This study aimed to establish a rat liver transplantation model with a donation after cardiac death and explore the technique and experience.Methods:there were 3 groups:group A(warm ischemia for 0 minute),group B(warm ischemia for 10 minutes),group C(warm ischemia for 20 minutes),and 40 pairs SD rats of each group.One hundred and twenty of rat model were performed liver transplantation by improved Kamada two-cuff technique.Postoperative 24h,72h,7-day survival rate and death causes were recorded and analyzed.Results:The 24 hours survival rate of recipients was 92.5%in group A,90.0%in group B and 92.5%in group C.The 72 hours survival rate of recipients was 90.0%,80.0%in group B and 77.5%in group C.The 7 days survival rate of recipients was 85.0%in group A,70.0%in group B and 57.5%in group C.There were no significantly differences of 24 hours and 72 hours survival rate in 3 groups(P>0.05).However,the survival rate were not the same in 3 groups(p =0<0.05).The survival rate of group C was significantly lower than that of group A(p =<0.0167).The cause of death was not sufficient for operation Within 24 hours after surgery.Death caused by biliary leakage,ischemic liver failure died after suargery 24-72 hours.After surgery 3-7 days,the cause of death was biliary complications,and with the extension of warm ischemia time,the incidence of biliary complications are higher.Conclusion(s):A stable rat DCD orthotopic liver transplantation model was established.The key to establish a stable DCD orthotopic liver transplantation model with a donation after cardiac death is to protect liver and bile duct function.and The difficulties are the anastomosis of suprahepatic vena cava and the shortening of the hepatic phase.Part?Relationship between Warm Ischemic Time and CaMK ?,Hepatocyte Apoptosis and Mitochondrial Apoptosis after Orthotopic DCD Liver Transplantation in RatsObjective(s):To investigate the relationship between different warm ischemia time of donor liver and CaMK ?,hepatocyte apoptosis and mitochondrial apoptosis after rat DCD orthotopic liver transplantation.Methods:Establish rat DCD liver transplantation model,group A(thermal ischemia Omin),group B(warm ischemia 10min),group C(warm ischemia 20min),six rats were randomly sacrificed in each group on day 1,3,and 7 after liver transplantation.The rate of hepatocyte apoptosis was measured by flow cytometry Annexin V-FITC/PI marker and the mitochondrial membrane potential was measured by flow cytometry JC-10 marker.,Quantitative determination of CaM mRNA and CaMK ?? mRNA in liver tissue was performed by using QRT-PCR.Western blot was used to detect CaM and CaMK ?? quantification in liver tissue,and Cyt C and AIF were quantified in liver cytoplasm.Results:At 3 days and 7 days after operation,the percentage of JC-10-labeled green fluorescent cells in hepatocytes was significantly higher in group C than in group A and B(P<0.05),and was higher in group B than in group A(P<0.05)..The early apoptosis rate,late apoptosis rate,and cell necrosis rate in the three groups at the same time point were higher in group C than in group B and group A,and in group B compared with group A(p<0.05).The expression of CaM,CaMK ??,Cyt C,and AIF had no significant difference between the groups one day after surgery.The C group was higher than the B group and the A group at 3 days and 7 days after operation(P<0.05),while the B group was higher than the A group.(P<0.05).Conclusion(s):In rat DCD orthotopic liver transplantation,as the time of the warm ischemia of the donor liver increase,the mitochondrial apoptosis and apoptosis rate increase gradually,The amount of expression of Cyt C and AIF in the cytoplasmic of hepatocytes and CaM,CaMK ? in liver tissue is gradually raised.It suggests that CaMK ? is associated with apoptosis and mitochondrial apoptosis after DCD liver transplantation in rats,and plays a key role in the hepatocyte apoptosis of mitochondrial apoptotic pathway after DCD orthotopic liver transplantation in rats.Part ?A preliminary study on the role of CaMK ? in apoptosis and mitochondrial apoptosis after DCD orthotopic liver transplantation in ratObjective(s):To further explore the role of CaMK ? in mitochondrial apoptosis and apoptosis after DCD orthotopic liver transplantation in rats.Methods:By setting up an DCD orthotopic liver transplantation model in rats for 10 min with warm ischemia,and targeting CaMK ?? gene and CaMK ??-shRNA lentivirus for transfection,the expression of CaMK? in hepatocytes was up-regulated and down-regulated.1B group(saline blank group),2B group(CaMK ?? overexpression empty vector group),3B group(CaMK ?? overexpression group),4B group(CaMK?? interference expression empty vector group),5B group(CaMK ??-interfering expression group).Thee rats were randomly sacrificed in each group on day 1,3,and 7 after liver transplantation.The rate of hepatocyte apoptosis was measured by flow cytometry Annexin V-FITC/PI marker and the mitochondrial membrane potential was measured by flow cytometry JC-10 marker.,Quantitative determination of CaM mRNA and CaMK ?? mRNA in liver tissue was performed by using QRT-PCR.Western blot was used to detect CaM and CaMK ?? quantification in liver tissue,and Cyt C and AIF were quantified in liver cytoplasm.Results:The percentage of JC-10-labeled green fluorescent cells,early apoptosis rate,late apoptosis rate,and necrotic cell rate of hepatocytes in the five groups were significantly higher than those in groups 1B and 2B(P<0.05),5B.The group was significantly lower than the 1B group and the 4B group(P<0.05),but there was no significant difference between the 1B,2B,and 4B groups(P>0.05).The expression of CaM,CaMK ??,c Cyt C,and AIF had no significant difference between the groups one day after surgery.The 3B group was significantly higher than the 1B and 2B groups at 3 days,7 days,and 30 days after operation(P<0.05).The difference between the 1B group and the 4B group was statistically significant(P<0.05),but there was no significant difference between the 1B,2B,and 4B groups(P>0.05).Concdusion(s):After DCD orthotopic liver transplantation in rats,lentivirus was injected from penis vein of rats for infection.This method is simple,safe,and effective.In the rat DCD orthotopic liver transplantation,after up-regulating the expression of CaMK ??in hepatocytes,the mitochondrial apoptosis and apoptosis rate were increased.Conversely,after interference with the expression of CaMK ? in hepatocytes,the mitochondrial apoptosis and the rate of apoptosis decreased.CaMK ? is involved in the regulation of hepatocyte mitochondrial apoptosis and apoptosis after DCD orthotopic liver transplantation in rats.It is concluded that if the CaMK ? signaling pathway is specifically blocked after DCD liver transplantation,the purpose of reducing hepatocyte apoptosis can be achieved.