Effects Of CaMK?? On Ischemia-reperfusion Injury In Rat DCD Liver Transplantation

Part One Construction and identification of rat CaMK?? gene lentivirus vectorObjective:To construct and identify the overexpression and interference of CaMK??gene lentiviruses of rat,which will provide a basis for further study on the effect of lentivirus-mediated interference of CaMK?? gene on the regulation of Ca2+/CaM/CaMKII signaling pathway and the effect of CaMK?? on ischemia-reperfusion injury in rat DCD liver transplantation.Methods:According to the mRNA sequence of rat CaMK?? gene,CaMK??gene was synthesized with whole gene,and the rat CaMK?? overexpression lentiviral vector and CaMK?? overexpression lentiviral control vector were constructed.According to the previous experiments of our group,the siRNA sequence with the highest interference efficiency was constructed into rat CaM??shRNA lentiviral vector and CaMK??shRNA lentiviral control vector.And we transformed them into E.coli TOP 10 competent cells and we verified them by sequencing.Subsequently,we transfected the lentivirus vector into 293T cells for packaging and virus titre were determined through absolute quantitative qPCR.Western Blot was used to detect the protein expression of CaMKIIy overexpression lentivirus vector in rats to confirm the validity of CaMK?? overexpression in rats.We transfected rat hepatocytes BRL-3A with different groups of lentiviruses,which were divided into normal saline control group(CON group),CaMK?? overexpression lentiviral control vector group(CON-mCaMKIIy group),CaMK?? overexpression lentiviral vector group(mCaMKIIy group),CaMK?? interference lentiviral control vector group(CON-shCaMKIIy group),and CaMK?? interference lentiviral vector group(shCaMK?? group).The expression of CaMK?? protein in each group was detected by Western Blot.Results:The lentiviral vector and its control vector of rat CaMK??overexpression of rat were successfu?? constructed,and the protein expression of rat CaMK?? overexpression lentiviral vector was detected by Western Blot.The lentiviral vector and its control vector of rat CaMK??shRNA were successfully constructed according to the siRNA sequence with the highest interference efficiency of our group.The titres of lentiviral vector of rat CaMK?? overexpression and lentiviral vector of rat CaMK??shRNA were 2E+9TU/mL which determined by absolute quantitative qPCR assay.After 48 hours of different lentiviruses transfection into rat hepatocytes BRL-3A,the protein expression of CaMK?? in mCaMK?? group was the highest,and that of CaMK?? in shCaMK?? group was the lowest.The protein expression of CaMK?? in mCaMK?? group was higher than that in CON-mCaMK?? group(P<0.01),and the protein expression of CaMK?? in shCaMK?? group was lower than that in CON-shCaMK??group(P<0.01).Conclusion:The lentiviral vector of rat CaMK?? overexpression was successfully conducted and expressed in 293T cells.The lentiviral vector of rat CaMK??shRNA was successfully conducted in 293T cells.The protein expression of rat CaMK?? overexpressing lentivirus in rat hepatocyte BRL-3A was significantly increased,while which of rat shCaMK?? interfering lentivirus in rat hepatocyte BRL-3A was significantly decreased.The CaMKIIy was used as a target gene to interfere with Ca2+/CaM/CaMK? signaling pathway,which will provide a research basis for the effect of CaMK?? on ischemia-reperfusion injury in rat DCD liver transplantation.Part Two Establishment of SD rat DCD orthotopic liver transplantation modelObjective:To establish a stable SD rat DCD orthotopic liver transplantation model and determine the optimal warm ischemic time for orthotopic liver transplantation model of DCD in SD rats in our subsequent experiments.Methods:In this experiment,the DCD orthotopic liver transplantation model in rats was established through controlling different warm ischemia time(warm ischemia 0 min,warm ischemia 15 min,and warm ischemia 30 min)with Kamada"two-cuff" method,and we determined the optimal warm ischemia time of animal model for this experiment through survival time and liver injury.Results:The survival rate of DCD orthotopic liver transplantation in rats at 0 min,15 min and 30 min of liver warm ischemia was 90%,80%and 60%,respectively.With the prolongation of warm ischemia time,liver injury became more serious.Liver function serum ALT,AST and TBIL gradually increased,and liver pathological sections showed that hepatocytes ranged from mild edema to punctate necrosis,then bridging necrosis to massive necrosis.Conclusion:In DCD orthotopic liver transplantation model in rats,the length of warm ischemia time is a key factor that directly affects the quality of donor liver.With the prolongation of warm ischemia time,liver function injury is gradually aggravated,which is consistent with pathological results.The survival rate of recipients decreased significantly with prolonged warm ischemia time after operation.By comparison,15 min of warm ischemia is an ideal time for the DCD orthotopic liver transplantation model in rats in further experiments,which can not only ensure the survival rate of the recipient after transplantation but also observe whether the intervention of lentivirus vector is effective for liver injury.Part Three The relationship between CaMKIIy and ischemia-reperfusion injury in DCD orthotopic liver transplantation in rats at different warm ischemia timeObjective:To investigate the relationship between CaMK?? and ischemia-reperfusion injury in DCD orthotopic liver transplantation in rats at different warm ischemia time.Methods:The DCD orthotopic liver transplantation model in rats with warm ischemia time of 0min,15min and 30min was established in part two.Twenty rats were sacrificed respectively in the three groups at 1 day,3 days and 7 days after liver transplantation.We used hepatocyte to measure Ca2+concentration and mitochondrial membrane potential energy through flow cytometry.Tunel method was used to measure hepatocyte apoptosis in liver tissue.Western Blot was used to detect the protein expression of CaM and CaMKIIy in liver tissue and that of AIF and Cyt C in liver cytoplasm.CaM mRNA and CaMKIIy mRNA in liver tissue was detected by QRT-PCR.The protein expression of CaM and CaMKIIy in liver tissue was detected by immunohistochemistry.We investigate the relationship between CaMKIIy and lschemia-reperfusion injury in DCD orthotopic liver transplantation in rats at different warm ischemia time.Results:The level of Ca2+ reached its peak at 1 day,decreased at 3 days,and increased gradually at 7 days after transplantation in the three groups.At the same time point,the concentration of Ca2+in 15 min and 30 min of warm ischemia group was higher than that in 0 min of warm ischemia group(P<0.01).The percentage of green fluorescent cells labeled with JC-10 increased with time,which suggested that the percentage of mitochondrial membrane potential energy decreased gradually.There was no statistical difference among three groups at 1day after operation.At 3 days after transplantation,the percentage of green fluorescent cells in warm ischemia 15 min group and 30 min group was higher than that in warm ischemia 0 min group(P<0.05).At 7 days after transplantation,the percentage of green fluorescent cells in warm ischemia 15 min group and 30 min group was higher than that in warm ischemia 0 min group(P<0.01).Tunel results showed the percentage of hepatocyte apoptosis in warm ischemia 30min group was higher than that in warm ischemia 0 min group(P<0.05)at 1 day after transplantation.There was no significant difference between warm ischemia 15min group and warm ischemia 0 min group.At 3 days after transplantation,the percentage of hepatocyte apoptosis in warm ischemia group at 15 min and 30 min was higher than that in warm ischemia group at 0 min(P<0.05).At 7 days after transplantation,the percentage of hepatocyte apoptosis in warm ischemia group 30 min was higher than that in warm ischemia 0 min group and the percentage of apoptotic cells in warm ischemia 30 min group was higher than that in warm ischemia 15 min group(P<0.05).The protein expression of CaM,CaMKIIy,AIF and Cyt C were measured by Western Blot,which increased with the prolongation of warm ischemia time(P<0.05,P<0.01).At the same time point,the protein expression in 15 min and 30 min of warm ischemia group was higher than that in 0 min of warm ischemia group.The trend of CaMmRNA?CaM??mRNA measured by QRT-PCR and the positive rates of CaM,CaMK?? measured by immunohistochemistry was almost the same.Conclusion:Warm ischemia time is the key factor affecting the quality of donor liver.After DCD orthotopic liver transplantation in SD rats,the longer ischemic time of donor liver was,the more serious of liver function injury was.The Ca2+concentration after transplantation increased.It was measured from molecular,protein and tissue levels that with the prolongation of warm ischemia time,the expression of CaM and CaMKIIy increased.CaMKIIy acted on mitochondrial membrane,the cell ratio of decreasing mitochondrial membrane potential energy increased.AIF and CytC were released from mitochondria and mitochondrial apoptosis increased,and hepatocyte apoptosis induced by mitochondrial apoptosis increased.Part Four The relationship between lentivirus-mediated CaMK? and ischemia-reperfusion injury in rat DCD orthotopic liver transplantationObjective:To investigate the relationship between lentivirus-mediated CaMKIIy and ischemia-reperfusion injury in rat DCD orthotopic liver transplantation.Methods:The DCD orthotopic liver transplantation model in SD rats were established with 15 min of warm ischemia time,for each transplanted rat,intraoperative injection with CaMKIIy overexpression and its control lentivirus(mCaMKII group,CON-mCaMKIIy group),CaMKIIy interference and its control lentivirus(shCaMKIIy group and CON-shCaMKIIy group)and the saline group(CON group)were carried out for transfecting into the transplanted liver through portal vein according to 1.0�108TU/mL.Twenty rats were sacrificed respectively at 1 day,3 days,and 7 days after transplantation.Serum was taken for liver function test.Hepatocytes were taken for flow cytometry to measure Ca2+concentration and mitochondrial membrane potential energy.Western Blot was used to detect the protein expression of CaM,CaMK??in liver tissue and that of AIF and Cyt C in hepatic cytoplasm.The expression of CaM mRNA and CaMKIIy mRNA in liver tissue was detected by QRT-PCR.The protein expression of CaM and CaMK?? in liver tissue was detected by immunohistochemistry.Ultrastructural changes in hepatocytes and mitochondria were detect by electron microscopy.Results:The survival rates at 1day,3 days and 7 days after transplantation in CaMK?? overexpression lentivirus group were 83.3%,78.3%and 70.0%,respectively.While the survival rates at lday,3 days and 7 days after transplantation in CaMK?? interference lentivirus group were 98.3%,93.3%and 88.3%,respectively,which suggesting that the interference of CaMK?? expression is a protective factor for liver damage after liver transplantation in rats,which can improve the survival rate of DCD liver transplantation in rats.The results of liver function showed that serum ALT and AST level were higher in the mCaMK?? group than those in the CON-mCaMKIIy group at all time points after transplantation,while serum ALT and AST level in shCaMK?? group were lower than those in CON-shCaMKIIy group(P<0.01).And there was no significant difference in serum TBIL among groups at 1 day after transplantation,the results were the same with serum ALT and AST level at 3 and 7 days after transplantation.The concentration of Ca2+ reached its peak one day after transplantation.There was no significant difference among the groups.It gradually decreased at 3 days after transplantation and increased again at 7 days after transplantation.The concentration of Ca2+in mCaMK?? group was higher than that in CON-mCaMK?? group at 3 and 7 days after transplantation.and that in shCaMKIIy group was lower than that in CON-shCaMKIIy group(P<0.01).The ratio of green fluorescent cells labeled with JC-10 was lower in shCaMKIIy group than that in CON-shCaMK?? group at 1 day and 3days(P<0.05,P<0.01),the ratio of green fluorescent cells was higher in mCaMK?? group than that in CON-mCaMK?? group at 7 days after operation,and the ratio of green fluorescent cells was lower in shCaMK?? group than that in CON-shCaMK?? group(P<0.01).The results of hepatocyte apoptosis were measured by Tunel method,which showed that the apoptosis rate of hepatocytes in mCaMK?? group was higher than that in CON-mCaMK?? group at 1 day,3 days and 7 days after transplantation,and the apoptosis rate of hepatocytes in shCaMK?? group was lower than that in CON-shCaMK?? group(P<0.01).The protein expressions of CaM,CaMK??,AIF were detected by Western Blot,at 1 day,the expressions in mCaMK?? group was higher than that in CON-mCaMK?? group(P<0.01,P<0.05),at 3 and 7 days,the expressions in mCaMK?? group was higher than that in CON-mCaMK?? group,and that in shCaMK?? group was lower than that in CON-shCaMK?? group(P<0.01).At 1 day,3 and 7 days after transplantation,the expression of Cyt C in mCaMK?? group was higher than that in CON-mCaMK?? group,and that in shCaMKIIy group was lower than that in CON-shCaMK?? group(P<0.05,P<0.01).The trend of expression of CaM mRNA,CaMK?? mRNA detected by QRT-PCR and positive rate of CaM and CaMK?? protein detected by immunohistochemistry were the same With that of Western Blot.Electron microscopy showed ultrastructural damage in the mCaMK??group,hepatocyte swelling and necrosis,mitochondrial swelling and cristae disappearing,while the damaged morphology of the shCaMK?? group was significantly improved,the hepatocytes were generally normal,and the mitochondria or hepatocytes were slightly swollen.Slight ultrastructural damage of liver tissue was observed from CON group,CON-mCaMKIIy group and CON-shCaMK?? group.The damage in mCaMK?? group gradually increased,and that in shCaMK?? group gradually decreased with time.Conclusion:The abnormal expression of CaMKIIy in DCD donor liver caused by ischemia-reperfusion injury can induce mitochondrial damage by activating Ca2+/CaM/CaMK? signaling pathway.Up-regulation of CaMK?? can increase the level of Ca2+and CaMK?? acts on mitochondrial membrane,which decreases the potential energy of mitochondrial membrane and increases the permeability of mitochondria,leading to mitochondrial swelling,rupture and apoptosis.The release of AIF and Cyt C of mitochondria into the cytoplasm can initiate mitochondria apoptosis and futher promote hepatocyte apoptosis;While down-regulation of CaMKIIy can decrease the level of Ca2+,mitochondrial membrane potential energy and AIF and Cyt C of mitochondrial release into cytoplasm,thereby reducing mitochondrial apoptosis and hepatocyte apoptosis.It has protective effect on liver function injury after DCD orthotopic liver transplantation in SD rats.Overexpression of CaMK?? plays a leading role in the mitochondrial apoptosis induced by ischemia-reperfusion injury in DCD donor liver.The specific interference with CaMK?? expression can specifically regulate Ca2+/CaM/CaMK? signaling pathway and effectively improve liver injury and postoperative survival rate of DCD orthotopic liver transplantation in SD rats.