Recombinant Adenovirus Vector Of Ad5-expression Of Galectin-9 In Rat Liver Transplantation Anti-rejection Experimental Study Of The Reaction

The Research on the Construction of Ad5-gaIectin-9 Vector and its Antirejection Effect in Rat Liver TransplantationPart One Establishment of Acute Rejection Model of Orthotopic Liver Transplantation in the RatObjective:To establish a stable acute rejections model of orthotopic liver transplantation in the rat, and observe the characteristics of the acute rejection, so that to offer a technical platform for further experimental studies.Materials and Methods:1 We underwent OLT skill training in closed population SD rats via two-cuff technique.2 Compared with the survival rate, complication and pathologic damage was observed in the two groups between DA-Lewis and Lewis-Lewis rats after liver transplantation.every 5 recipients were sacrificed respectively at day 4,7,10 after OLT to detect the changes of serum biochemistrp ALT (alanine aminotransferase,ALT) and TBIL(total bilirubin, TBIL)and the grafts histology. the other 5 recipients were left in each group to observe the animal survival.Results: 1 The survival time of the rats were no more than 72 hours in the skill training period, but the experiences of pre-surgery and post-surgery management were accumulated.2 ALT values in DA-Lewis group was progressive advanced but gradually stepped down in Lewis-Lewis group, There was significantly difference (P<0.01) in two groups at the same tome.3 Compared with Lewis-Lewis group, TBIL values in DA-Lewis group was progressive advanced at day 7,10 (P<0.01).4 In DA-Lewis group, the histological manifestations of the grafts were obvious rejection at day7, and became more sever at day 10. The Banff scale of grafts in DA-Lewisr group wasⅡtoⅢgrade rejection but in Lewis-Lewis group was 0 grade rejection at day 10.Conclutions:1 The establishment a stable of orthotopic liver transplantation model in the rat has great difficulties, and there were plenty of influencing factors. The surgery was success or not determined on masterly microsurgical technique, consummate pre-and post-surgery management and patient operation2 There were notable and stable characteristics of acute rejections in the DA-Lewis rats orthotopic liver transplantation model, so that further researches could use of this model. Part Two Cloning of rat galectin-9 full-length cDNA and construction of its recombinant adenovirus vectorObjective:To clone rat galectin-9 full-length cDNA and procure recombinant adenovirus granule containing rat galectin-9 gene.Methods:The rat galectin-9 gene was amplified by RT-PCR method from rat liver tissue and inserted orientationally into plasmid pDC316 digested by restriction endonucleases NotⅠand HindⅢ. The recombinant pDC316-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then cotransfected with rescue plasmid PBHGloxΔE1.3Cre into 293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene(Ad5-galectin-9) was generated by site-specific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in 293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay.Results:Construction of pDC316-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 IU/ml.Conclusions:Recombinant adenovirus vector containing rat galectin-9 gene(Ad5-galectin-9)was successfully constructed, which provides the foundation of further researching function of galectin-9 gane.Part Three Effects of Ad5-galectin-9 on preventing allograt from acute rejection following rat orthotopic transplantationObjective:To study the mechanism and protective role of Ad5-galectin-9 in acute rejection following rat orthotopic liver transplantation.Methods:Orthotopic liver transplantation was performed in male inbred DA rats to Lewis rats. Ad5-galectin-9 or control empty vector was deliver to graft during the preservation.5 groups were designed in the study, Group Axeceived unmodified grafts; Group B:received hepatic grafts transduced empty vector; Group C:received heptic graft with intragastric administration of CsA at a dose of 10mg/kg; Group D:received hepatic grafts transduced Ad5-galectin-9:Group E: Ad5-galectin-9+CsA(5mg/kg). all of groups were divided into four subgroups. Recipients of a subgroup were sacrificed on day 4,7 or 10 respectively, serum levels of ALT,AST and TBIL were measured, Part of the liver tissues were made into paraffin-embedded specimens to detect rat liver histological change and grade RAI(Banff scheme):apoptosis of hepatocyte and Th1 were monitored by FCM; the serum levels of IL-2,IL-4,IL-10 and IFN-γware tested by ELISA; the galectin-9 gene products of allograft was detected by Western Blotting. Results:Compared with group A and D, survival time of recipients in group C,D,and E was significantly prolonged (P<0.05). The level of ALT,AST and TBIL in group A and B were rapid advanced but gradually recoveried in group C,D and E. The level of IL-2 and IFN-yin group A and B were higher than group C,D and E(P<0.01), and group C vs group D and E(P<0.05). The level of IL-4 and IL-10 in group A and B were lower than group C,D and E at the same time (P<0.01). compared with group A and B, RAI(according to Banff scheme) was significantly decreasd(P<0.05). As compared with group A and B, the apoptosis rate of hepatocyte decreased obviously after ROLT in group C,D and E(P<0.01).Conclusion:Doner pretreatment with Ad5-galectin-9 preventing allografts from acute rejection in ROLT. Subtherapeutic half dose of CsA act synergistically with Ad5-galectin-9 to significantly inhibit acute rejection of allograft. Ad5-galectin-9 preventing allograts from acute rejection may be associated down-regulated the expression Thl cytokine.Galectin-9 may be a novel therapeutic drug in transplant medicine.