| Co-Investigator(Kenkyū-buntansha) |
OKUYAMA Torayuki Dept. of Exp. Surgery, National Children's Medical Research Center Deputy Director, 小児医療研究センター・先天異常研究室, 室長 (40177192)
LI Xiao-Kang Dept. of Exp. Surgery, National Children's Medical Research Center, Research Reader, 小児医療研究センター・実験外科研究室, 研究員 (60321890)
AMEMIYA Hiroshi The Head of National Children's Medical Research Center, 小児医療研究センター, センター長 (80009563)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Hepatocyte transplantation has been proposed for a potential therapeutic method to treatment liver failure and inherited hepatic disorders, however, transplanted hepatocytes are difficuft to reconstruction the fiver under normal condition. The purpose of this study is to investigate how to modulate the recipient liver condition and promote the repopulation of mlce liver after Fas-resistant hepatocyte transplantation. The hepatocytes, in a number of 1×106 wereisolated from male MRL, lpr/lpr mice which display the mutant of the Fas antigen, and transplanted to the female MRL, +/+, wild type mice by intrasplenic injection. An agonist anti-Fas antibody, 0.15mg/kg was administered intravenously at 24hr after hepatocytes transplanted. Then the doses of administration antibody were increased to 0.3mg/kg, at 1 week and 0.6mg/kg, at 2 weeks, respectively. The liver specimens were taken at different time points after transplantation for examination the fiver pathological and immune histological
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changes. The hepatocyte reconstruction was determined by real time quantitative PCR assay, using the primers and probe for sry gene. The pathologic changes of the recipient liver after nonlethal dose of ants-Fas antibody treatment showed massive apoptotic hepatocytes were confirmed. The male transplanted Fas-resistant hepatocytes were selective reconstruction as much as 6.9% of the normal fema(e recipient liver in ants-Fas antibody treatment group at 3 month. tn contrast, the reconstruction of the transplanted cell were lower than 0.1% in non-treatment group.
These findings demonstrate that repeated apoptoic-induce challenges shift environment of recipient liver to regenerative condition is useful to promote the transplanted hepatocytes reconstruction in the mice fiver.
Cell-mediated cytotoxicity may be involved in delayed and/or chronic xenograft rejection in which apoptosis is induced in the grafted celts via the Fas/Fas-figand (FasL) and perforin/granzyme pathways. One barrier to the potential use of xeonogenic grafts for human may be Fas/FasL-mediated apoptosis, which would be blocked by the gene expression of cytokine response modifier A (CrmA), a cowpox virus gene product. The purpose of this study is to explore whether crmA is an effective candidate gene for inhibiting apoptosis in an in vitro model of xenograft rejection, using Fas-expressing
non-primate cells cultured with a soluble recombinant human FasL (sFasL). A recombinant adenovirus vector expressing CrmA (AxCALNLCrmA) was successfully generated with a ere-mediated switching system. PK15 cells, derived from a porcine kidney and infected with AxCALNLCrmA and/or AxCANCre at a multiplicity of infection (MOl) ranging from 0.1 to 100, were cultured with human sFasL derived from KFL74.18, a human FasL-overexpressed cell fine.
The gene-expression level of the PK15 cells was confirmed by CrmA-immune staining. Approximately 70% of the control PK15 cells showed induced apoptosis when cultured with sFasL, . In contrast, the apoptosis was dramatically reduced in crmA-gene-transduced PK15 cells. The inhibitory effect of apoptosis increased with an increase in the infection dose of AxCANCre. In addition, the activity of caspases 3 and 8 was significantly inhibited in the crmA-transduced cells. These results indicate that CrmA is an effective gene product for inhibiting Fas/FasL-mediated apoptosis, which suggests the potential therapeutic use of its gene-transduction to protect against graft damage due to delayed and/or chronic xenograft rejection. Less
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