| Co-Investigator(Kenkyū-buntansha) |
ブルーム エダ T. 米国食品医薬品管理局, リサーチセンター, 室長
YAMAUCHI Akira Kyoto University, Graduate School of Medicine, Assistant, 医学研究科, 助手 (00291427)
INAMOTO Takashi Kyoto University, College of Medical Technology, Professor, 医療技術短期大学部, 教授 (10135577)
BLOOM Eda T FDA, CBER, Lab. Chief
|
|---|
| Budget Amount *help |
¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1997: ¥4,900,000 (Direct Cost: ¥4,900,000)
|
|---|
| Research Abstract |
We investigated the availability of ilterleulkin (IL) -12 for the gene therapy of hepatoma as a joint research with Dr,. Eda T. Bloom in FDA in the United State. We made a rat cirrhotic liver with use of thioacetamide as a in vivo model to evaluate the efficacy of IL-12 for the therapy of human hepatoma. We implanted AH66F cells, syngenic hepatoma cells, beneath the capsule of rat liver and performed the intra-peritoneal administration of IL-12. Tumor weight measured 7 weeks after administration was 0.7g, which was significantly lower than control (2.2g). On the contrary, IL-12 has an adverse effect to the liver when administrated in vivo. To reduce the dose of IL-12, we examine the synergistic effect of IL-12 and IL-18, which can induce IL-12 receptors on NK or T cells. IL-18 enhanced IL-12 mediated cytotoxic activity of separated human NK cells. We investigated the intracellular optimal microenvironment where NK cells can have high cytotoxic activity. The citotoxic activity of NK cel
… More
ls from cirrhotic rat liver or resected human liver bearing cirrhosis and hepatoma was significantly impaired, N-acetylcystein (NAC), the precursor of glutathione, resored their activity up to normal level in vitro. Further more, in vivo administration of both IL-12 and NAC suppressed the progression of tumors implanted in cirrhotic rat liver (0.3g). On the other hand, we planned to introduce IL-12 gene to liver cells. IL-12 gene (p40 and p35 were provided by Genetic Institute, USA. As preliminary assay, β-galactosidase gene with chicken β-actin promoter (LacZ) and FITC labeled oligodeoxynucleotide (FITC-ODN) were prepared. HVJ-liposome or HVJ-cationic liposome was prepared as follows ; dried lipids containing phosphatidylserine or DC cholesterol were mixed for liposome or cationic-liposome, respectively. These suspensions were incubated with DNA or FITC-ODN and inactivated HVJ. Hep3B and Huh7 (human hepatoma cell line) were transfected with these liposome complexes. These were directly injected into Wister rat liver in vivo. In order to detect the cellular uptake of FITC-ODN, transfected cells or livers were examined by fluorescent microscopy. X-gal staining was performed 72 hours after the injection of HVJ-liposome containing LacZ. After transfection of HVJ-liposome containing LacZ, only 2 or 3% of the Hep3B and Huh7 cells were positive in X-gal staining. However, fluorescence was detected in almost all cells even at 30 minutes after transfection of HVJ-cationic liposome containing EITC-ODN, especially 50-60% of nucleus of cells were accumulated. X\gal staining of rat liver transfected with HVJ-liposome containing LacZ revealed that 57% cells of hepatocytes were positive. In contrast, fluorescence was observed only in non-parenchymal cells in vivo after transfection of HVJ-cationic liposome containing FITC-ODN, probably due to interference by non-parenchymal cells. Although IL-12 expression was not detected by Western blotting nor ELLISA by now, the combination therapy of IL-12 by HVJ-liposome method with IL-18 or NAC may mediate the optimal cytotoxic activity of liver lymphocytes. Less
|
