The Experiment Study On Inducement Of Rat Arterized Partial Liver Transplantation Tolerance By Constructing Adeno-associated Virus Contained CTLA4Ig

Part â… Significance of arterial reconstruction on liver grafts after partial orthotopic liver transplantation in ratsObjectiveThe purpose of this study was to investigate the significance of arterial reconstruction on liver grafts after partial orthotopic liver transplatation in rats.MethodsA total of 96 rats were randomly divided into 2 groups: partial orthotopic liver transplantation without arterial reconstruction group(POLT) and partial orthotopic liver transplantation with arterial reconstruction group. Liver function, histology and proliferate index(PI) by Flow Cytometry were detected 1, 2, 4 and 7 days after surgery respectively.ResultsAlanine aminotransferase(ALT) and total bilirubin(TB) began to elevate from the first day after surgery. ALT and TB in POLT group on fourth day were higher significantly than those in APOLT group(P<0.05).Histological findings indicated that more diploid and polyploid hepatocytes could be seen in APOLT group. PI in APOLT group on the second and fourth day were higher than those in POLT group significantly [(33.81 ± 3.45)%, (35.33 ± 2.52) vs (23.08 ± 4.66), (29.79 ±ConclusionArterial reconstruction can improve liver function of liver grafts after POLT, alleviate the histological changes and promote the regeneration of liver grafts.Part â…¡Construction of adeno-associated virus vector expressing CTLA4Ig and its expression in the transplanted liver allograftsObjectiveOur purpose of this study was to construct the recombinant adenoassociated virus vector pSNAV expressing CTLA4Ig and to demonstrate its expression in transplanted liver allografts. Using the adeno-associated virus vector expressing CTLA4Ig, we mignt obtain the long term inhibitive effect of CTLA4Ig.MethodsAfter AAVCTLA4Ig and PUC18 being cut with BamH I respectively, CTLA4IgcDNA was inserted into plasmid PUC18 by T4DNA ligase and PUC18-CTLA4Ig was constructed. The obtained PUC18-CTLA4Ig and pSNAV being cut with Kpn I and EcoR I , CTLA4IgcDNA was inserted into plasmid pSNAV to construct recombinant vector pSNAV- CTLA4Ig, which was transfected into BHK-21 packaging cells by lipofectine-mediated transfectoin. Then the BHK-21 cell line was infected with HSV1-rc to produce a large amount of pSNAV- CTLA4Ig. The specificity of expressed product was identified by digestion with BamH I , PCR and sequence determination. The titer of virus was detected. The product was infused into rats' liver allografts via portal vein and its expression in the transplanted livers was detected immunohistochimistry.ResultsRecombinant adenoassociated virus vector pSNAV-CTLA4Ig was generated and purifed into 8.5*10¹¹/ml. Agarose gel analysis of PCR products indicated the presence of CTLA4Ig. Digestion with BamH I and sequence determination confirmed pSNAV-CTLA4Ig was constructed. Expression of CTLA4Ig in the transplanted livers. was detected successfullyConclusionPrepared pSNAV-CTLA4Ig is constructed correctly and can express CTLA4Ig effectively. Besides this, it can express CTLA4Ig in the rats' liver allografts. It may be used in the study of transplant tolerance. Part â…¢Effect of adeno-associated virus vector encoding CTLA4Ig on the transplanted rat liver allograftsObjectiveTo investigate the immunological inhibition effects of CLTA4Ig on on inducement of rat arterized partial liver transplantation tolerance by constructing adeno-associated virus contained CTLA4IgMethodsOLT on rat was established by using "the two cuff technique". The recipients were divided into the following 5 groups randomly: SD-SD group(n=16): no treatment after livertransplantaion; control group(n=16): no treatment after livertransplantaion; pSNAV-LacZ Group (n=16): liver-grafts were flushed with 1*1011 v.g./ml pSNAV-LacZ 1ml via portal vein; CsA treatment group(n=16): the recipients were treated with CsA 8mg/kg/d via i.p. for 7 days; pSNAV-CTLA4Ig treatment group: liver grafts were flushed with 1*1011 v.g./ml of pSNAV-CTLA4Ig 1ml viaportal vein. Blood, spleen and liver samples were collected on 7 days after OLT for cytokines concentration, quantitative PCR for cyto...