| Project/Area Number |
15390381
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | Nagasaki University |
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Principal Investigator |
KANEMATSU Takashi Nagasaki University, Graduate School of Biomedical Sciences, Transplantation and Digestive Surgery, Professor, 大学院・医歯薬学総合研究科, 教授 (40128004)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASHITA Yujo Nagasaki University, Graduate School of Biomedical Sciences, Transplantation and Digestive Surgery, Assistant Professor, 医学部・歯学部附属病院, 助手 (20372774)
EGUCHI Susumu Nagasaki University, Graduate School of Biomedical Sciences, Transplantation and Digestive Surgery, Assistant Professor, 医学部・歯学部附属病院, 助手 (80404218)
TAKATSUKI Mitsuhisa Nagasaki University, Graduate School of Biomedical Sciences, Transplantation and Digestive Surgery, Assistant Professor, 医学部・歯学部附属病院, 助手 (80380939)
蒲原 行雄 長崎大学, 医学部・歯学部附属病院, 助手 (50325643)
古井 純一郎 長崎大学, 大学院・医歯薬学総合研究科, 助教授 (30264229)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥10,700,000 (Direct Cost: ¥10,700,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥7,400,000 (Direct Cost: ¥7,400,000)
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| Keywords | hepatocyte isolation / banking / gene transfer / hepatocyte transplantation / Decoy receptor3 / 肝細胞バンク / 凍結保存 / 肝細胞保存 / 細胞バンク / Decoy Receptor 3遺伝子導入 |
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| Research Abstract |
We cinfirmed that the isolation of hepatocytes from resected human liver tissues in the clinics. In addition, small hepatocytes were also isolated from the fraction of non-parenchymal hepatocytes as described by Mitaka et al. We characterized these cells and investigated the possibility of gene therapy.
1)Isolation of matured hepatocytes and small hepatocytes
Liver tissues digested by collagenase were filtrated and centrifuged at 50xg. As a result, matured hepatocytes were precipitated, however, small hepatocytes located in the supernatant fraction.
Result ; Small hepatocytes that could grow for more than 3 months were obtained which produced alpha1 anti-trypsin continuously.
2)Optimal ingredients of cryopreservative solution for isolated human hepatocytes
Various type of cryopreservatice solution including ; a)Cell banker, b)10%DMSO+UW, c)10%DMSO+10%FBS+UW, d)10%DMSO+FBS, e)UW were compared.
Results ; The highest viability (60%) of freezing and thawing hepatocytes were observed in 10%DMSO+UW group. However these freeze-thawed hepatocytes tended to lose adhesive capacity to the cell culture dishes, suggesting that the integrities of these cellular membranes were deteriorated.
3)HVJ-envelope vector mediated gene transfer into hepatocytes
We initially investigated the efficacy of the conventional HVJ-liposome for gene transfer into hepatocytes in both rats and pigs. Based on these results, we analyzed the transfection efficiencies of HVJ-envelope vector into the hepatocytes by using LacZ and luciferase reporter genes. Also histological and biochemical examination of these transfected organs in the animals were performed.
Results ; Subcapsular injection of the reporter genes shoed the highest transfection efficiency compared with portal vein injection. Other organs were not damaged by this transfection process according to the findings of histology.
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