| Objectives:1.Use the "two-cuff’ method to construct a SD rat cardiac death donor liver transplantation model,construct HO-1 overexpression and HO-1 shRNA recombinant adeno-associated virus and transfect the rat,recombine the HO-1 gene in vivo,and induce Liver HO-1 specific overexpression or expression interruption,to explore the effect of adeno-associated virus preconditioning SD rats with HO-1 expression on the survival rate of rats after liver transplantation from heart-dead donors;2.Cardiovascularization of SD rats After liver transplantation from dead donors,the changes in the expression of HO-1 over time after the transplanted liver undergoes ischemia-reperfusion injury for different lengths of time were analyzed,which proves that the expression of HO-1 after liver transplantation from dead cardiac donors is time-dependent,And further explore the mechanism of liver function damage after liver transplantation from a dead heart donor;3.Use HO-1 overexpression and HO-1 shRNA recombinant adeno-associated virus to transfect SD rats to induce specific overexpression or interruption of liver HO-1 expression,and to explore the intervention of adeno-associated virus pretreatment in SD rats with donor liver HO-1 Express the protective mechanism of liver transplantation after liver transplantation from a heart-dead donor.Methods:1.Select 280～300g,8～10-week-old SD rats,and store the donor liver under cold perfusion at 4℃ after 10 minutes of cardiac arrest.The cold storage time is 4h.The"two-cuff" method is used to establish the heart-dead donor liver.Transplant the model.2.Constructing HO-1 overexpression and HO-1 shRNA recombinant adeno-associated virus and transfecting 150-180g,5-6 week old SD rats via tail vein injection,and measuring the transfection efficiency in vivo by RT-qPCR after 21 days of rearing.3.After 6h,24h,72h,and 168h after ischemia-reperfusion injury of a heart-dead donor liver transplantation,the protein HO-1,SIRT1,pro-caspase-1,p22,full-GSDMD,The expression of cleaved-N-GSDMD.Immunohistochemistry was used to detect the expression of NLRP3 in paraffin specimens.Automatic biochemical analyzer detects serum ALT and AST and evaluates liver function.HE staining of paraffin sections was used to observe the degree of pathological damage of liver tissue.ELISA detects the content of IL-1β and IL-18 in liver tissue.Flow cytometry detects the expression level of ROS in liver cells and evaluates the degree of oxidative stress.4.HO-1 overexpression and HO-1 shRNA recombinant adeno-associated virus were transfected into donor rats,and the donor rats were transplanted with heart-dead donor liver.The 6h time point of postoperative ischemia-reperfusion injury was selected,and RT-qPCR and Western blot were used.Detect the expression level of HO-1 in liver tissue.The expression of SIRT1,pro-caspase-1,p22,full-GSDMD,and cleaved-N-GSDMD were detected by Western blot.Immunohistochemistry was used to detect the expression of NLRP3 in paraffin specimens.Automatic biochemical analyzer detects serum ALT and AST and evaluates liver function.HE staining of paraffin sections was used to observe the degree of pathological damage of liver tissue.ELISA detects the content of IL-1β and IL-18 in liver tissue.Flow cytometry detects the expression level of ROS in liver cells and evaluates the degree of oxidative stress.5.Use statistical software SPSS24.0 for data analysis.Using Kalplan-Meier survival analysis statistical method;measurement data in the form of x-±s,and multiple sets of measurement data were compared using ANOVA,advanced test of homogeneity of variance,homogeneity of variance is further using LSD test;variance missing Dunnetts T3 test is used.P<0.05 considered the difference to be statistically significant.The statistical graph was drawn with GraphPad Prism 8.0.Results:1.HO-1 overexpression,HO-1 shRNA recombinant adeno-associated virus transfected into SD rats,the difference of HO-1 gene transcription level was statistically significant(P<0.05).2.The SD rat heart death donor liver transplantation model was successfully constructed,and the difference in survival time obtained after HO-1 overexpression and HO-1 shRNA recombinant adeno-associated virus transfected into the donor rat was statistically significant(P<0.05).3.The expression of HO-1 is time-dependent with ischemia-reperfusion injury after DCD liver transplantation,and the increase is most obvious in the recent period(6h and 24h).Compared with the control group,the difference is statistically significant(P<0.05),While the long-term(72h and 168h)gradually decreased,although the expression was still higher than that of the control group,the difference from the recent period was also statistically significant(P<0.05);the expression of ROS,ALT and AST in the short-term after ischemia-reperfusion injury Significantly higher than the control group(P<0.05),but gradually decreased in the long-term;HE staining showed that the recent tissue necrosis was severe,and the Suzuki score was statistically significant compared with the control group and the long-term difference(P<0.05);Simultaneous western blot detection of SIRT1,pro-caspase-1,p22,full-GSDMD,cleaved-N-GSDMD,SIRT1 expression trend is consistent with HO-1,pro-caspase-1,p22,full-GSDMD,cleaved-N-GSDMD increased significantly in the short term,but decreased in the long term.Immunohistochemical detection of NLRP3 found that the expression level of cleaved-N-GSDMD was the lowest in the recent 6h,and then showed a gradual decrease from high over time.4.HO-1 overexpression and HO-1 shRNA recombinant adeno-associated virus were transfected into donor rats,and the donor rats were subjected to heart-dead donor liver transplantation.At 6 hours after the postoperative ischemia-reperfusion injury,compared with the HO-1 interference expression group ROS,IL-1β,IL-18,ALT,and AST in the HO-1 overexpression group were significantly reduced(P<0.05),and the expression of p22 and cleaved-N-GSDMD were also significantly inhibited(P<0.05);at the same time,HE The staining Suzuki score confirmed that the ischemia-reperfusion injury after liver transplantation in the HO-1 overexpression group was significantly less than that in the HO-1 interference expression group(P<0.05);immunohistochemical detection revealed that the HO-1 overexpression group had a higher expression of NLRP3 The control group decreased significantly,but the expression of NLRP3 in the HO-1 interference expression group was lower than that in the HO-1 overexpression group.This may be caused by increased necrosis and no normal liver tissue structure.Conclusions:1.The "two-cuff" method was used to successfully construct a SD rat heart-dead donor liver transplantation model.2.Pyroptosis is one of the mechanisms of ischemia-reperfusion injury after liver transplantation from a dead heart donor,and it is the most serious in the early stage of reperfusion,and then gradually relieved.3.Preconditioning the donor can make HO-1 highly expressed,it can inhibit the occurrence of hepatocyte pyroptosis through the SIRT1-mitochondrial pathway,thereby reducing the ischemia-reperfusion injury of the transplanted liver of the SD rat cardiac death donor. |
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