| Co-Investigator(Kenkyū-buntansha) |
RII Kou National Children's Medical Research Center, Department of Experimental Surgery & Bioengineering, Researcher, 小児医療研究センター・実験外科生体工学部, 研究員 (60321890)
KIMURA Hiromitsu National Children's Medical Research Center, Department of Experimental Surgery & Bioengineering, Chief Researcher, 小児医療研究センター・実験外科生体工学部, 室長 (80115477)
ENOSAWA Shin National Children's Medical Research Center, Department of Experimental Surgery & Bioengineering, Chief Researcher, 小児医療研究センター・実験外科生体工学部, 室長 (40232962)
NAKAJIMA Hiroo Kyoto Prefectural University of Medicine, Second Department of Surgery, Assistant, 第2外科, 助手 (70275212)
SHINOMIYA Takahisa National Children's Medical Research Center, Department of Experimental Surgery & Bioengineering, Division of Research Promotion, Chief Researcher, 小児医療研究センター・実験外科生体工学部, 室長 (30196414)
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| Research Abstract |
An adenovirus vector AxCALNFasL was constructed in order to transduce a gene for Fas-Ligand(FasL), requiring cotransfection of Cre recombinase-expressing vector AxCANCre for its expression. When intravenously injected AxCALNFasL, rat liver expressed FasL by co-administration of AxCANCre in a dose-dependent manner. The FasL-expressed livers survived significantly in the allogeneic recipients. However, a high level of FasL-expression induced animal death due to fulminant hepatitis. Furthermore, we successfully generated AxCALNLCrmA, a recombinant adenovirus expressing CrmA-gene with a Cre-mediated switching cassette. When allografted, the CrmA-expressed livers were resistant to rejection response in the recipients, resulting in marked prolongation of recipient survival. The formation of active caspase-3 was markedly inhibited in the Crm A gene-transfected hepatocytes. In addition, CTLA4Ig, a soluble recombinant fusion protein that contains the extracellular domain of CTLA4 and Fc portion
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of IgG1, strongly adheres to the B7 to block CD28-mediated costimulatory signals and inhibits in vitro and in vivo immune responses. We intravenously injected the adenovirus vector containing CTLA4Ig cDNA(AxCAhCTLA4Ig)to the heartgrafted rats immediately after grafting. The serum level of CTLA4Ig reached to maximum at 51-93μg/ml 3 to 7 days after gene-transfection and declined after 14 days, although detectable levels were observed up to 49 days. The graft survival time was significantly prolonged in the gene-transfected recipient rats. Down-regulation of IL-2 and IFNγ mRNA and persistence of IL-4 and IL-10 transcripts were observed in the graft-infiltrating cells. Peri-operative administration of FTY720, a new immunosuppressant inducing lymphocyte apoptosis, in combination with the CTLA4Ig gene-transfection resulted in synergistic prolongation of graft survival, and 33% of recipients acquired transplantation tolerance. We detected CTLA4Ig protein in the sera 49 days after grafting ; its levels were always hiher in the combination group than in the AxCAhCTLA4Ig-alone group. Thus, FTY720 would be highly effective for enhancing the CTLA4Ig expression by adenovirus-mediated gene transfer. Less
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