The Study On The Protective Effect Of ENOS Gene Transfer On IRI Of Rat Small-for-size Liver Transplantation

Part One Construction of Recombinant Adenovirus of eNOSObjective: To construct the recombinant adenovirus vector containing eNOS gene.Methods:(1) The recombinant adenovirus vector Ad-eNOS using gene sequencing.(2) The recombinant adenovirus vector Ad-eNOS by liposome mediated transfection293cells packaged into viral particles, using eNOS gene primers for identification of PCRsynthesis.(3)By measuring the absorbance of the virus titer was calculated and packed.Results:(1) By DNA sequencing, the Ad-eNOS gene sequence is consistent withGeneBank.(2) Identification of recombinant adenovirus Ad-eNOS packing is successfulby Real-time PCR.(3) The virus titer was9.35×109PFU/ml by the determination of virustiter.Conclusion: To construct the recombinant adenovirus vector Ad-eNOS by usingHEK293cells.To obtain recombinant adenovirus Ad-eNOS.Part Two Protective effects of eNOS gene transfer onhypoxia-reoxygenation injury in hepatocellular L02Objective: To investigate the protective effect of eNOS gene transfer on hypoxia-reoxygenation injury in hepatocellular L02.Methods:Using the L02liver cells, in the hypoxia model (including95%N2+5%CO2、PO2≤4Kpa) and reoxygenation model (85%O2+15%CO2，PO2≥13Kpa), constructinghypoxia-reoxygenation model, The experiments were divided into three groups:inexperimental group treated with Ad-eNOS; in control group and in normal group with RPMI-1640medium. The experimental group and the control group in hypoxia-reoxygenation model, in normal group were cultured in5%CO2, saturated humidityincubation box. The contents of ALT and NO was detected in the medium, the expressionof eNOS gene was detected by Real-time PCR, the expression of eNOS protein wasdetected by Western-blot, apoptosis cell was detected by FCM.Results: In experimental group, the content of ALT (26.26±3.78u/l) lower than thatof in control group (48.42±5.31u/l)(P <0.05), but higher than that in normal group(17.20±2.64u/l)(P <0.05); and the content of NO in experimental group (18.89±3.30umol/L) was significantly higher than in control group (6.44±2.11umol/L) and innormal group (8.85±2.40umol/L)(P <0.05); Real-time PCR and Western-blot resultsshowed that the expression of eNOS in experimental group in the gene and protein levelswere significantly higher than in control group and in normal group (P <0.05); FCMshowed that in experimental group of L02liver cell apoptosis was significantly lower thanin control group (P <0.05), and in normal group L02apoptosis of hepatic cells rarely.Conclusion:(1) Ad-eNOS gene was transfected into the L02liver cells, the highexpression of eNOS gene and protein;(2) eNOS gene transfer can reduce cell apoptosis onhypoxia-reoxygenation injury in hepatocellular L02;(3) eNOS gene transfer could haveprotective effect on hypoxia-reoxygenation injury in hepatocellular L02by rising the NOpathway.Part Three To establish the model of small-for-size liver transplantationin ratsObjective: to establish the rat model of small-for-size liver transplantation bymodified two cuff method.Methods: Based on the Kamada two cuff technique, improvement of the ratorthotopic liver transplantation in perfusion, repair, treatment with liver operation and perioperation period, to establish a stable rat orthotopic liver transplantation model; on thisbasis to establish the small-for-size liver transplantation in rat model. Results: By comparison of the three phase, the formal experimental group in theoperation time, anhepatic phase, the success rate of operation, complication were obviouslyincreased.Conclusion:(1)The modified two cuffed technique has the advantages of longsurvival time, short anhepatic phase operation, high success rate, which is an idealoperative method of orthotopic liver transplantation in rats;(2) Based on the stable modelof orthotopic liver transplantation in rats establishing of small-for-size liver transplantationin rats model is helpful to improve the stability of the model.Part Four The protective effect of eNOS gene transfer on IRI of ratsmall-for-size liver transplantationObjective: To investigate the protective effect of eNOS gene transfer on IRIsmall-for-size liver transplantation in rats.Methods:12SD rats were randomly divided into two groups (n=6) for small-for-sizeliver transplantation.The control group were intraperitoneal injection of donor with emptyvector, the experimental group with Ad-eNOS donor. After36hours small-for-size livertransplantation is operated. Six hours after reperfusion, the recipients were sacrificed, NOwere measured, TNF-α、macrophage levels were detected by immunohistochemistry.Apoptosis of the hepatocytes was analyzed by TUNEL and FCM; the expression of eNOSgene was detected by Real-time PCR, the expression of eNOS protein was detected byWestern-blot.Results:(1) In experimental groups ALT, AST, LDH were significantly lower thanin the control group; apoptosis rate was significantly lower than in control group;(2) thecontent of NO in liver tissue in experimental group was significantly higher than in controlgroup;(3) Real-time PCR and Western-blot results showed that in experimental groupeNOS at the level of gene and protein were significantly higher than in control group;(4)Immunohistochemistry showed that TNF-α and macrophages high expression in controlgroup and low expression in experimental group. Conclusion: eNOS has a protective effect on IRI of small-for-size livertransplantation in rats, and its mechanism may be related to the down-regulation of TNF-α,inhibiting the macrophage infiltration.