Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants |
|Research Institution||Sapporo Medical University |
HIRATA Koichi Sapporo Medical University, 1st Department of Surgery, Professor, 医学部, 教授 (50136959)
MITAKA Toshihiro Sapporo Medical University, Department of Pathophysiology Cancer Research Institute, Assistant Professor, 医学部, 教授 (50231618)
KATSURAMAKI Tadashi Sapporo Medical University, 1st Department of Surgery, Assistant Professor, 医学部, 助教授 (50253993)
古畑 智久 札幌医科大学, 医学部, 講師 (80359992)
MIZUGUCHI Toru Sapporo Medical University, 1st Department of Surgery, Assistant Professor, 医学部, 講師 (30347174)
本間 敏男 札幌医科大学, 医学部, 助手 (30315494)
KIMURA Yasutoshi Sapporo Medical University, 1st Department of Surgery, Instructor, 医学部, 助手 (80311893)
TSURUMA Tetsuhiro Sapporo Medical University, 1st Department of Surgery, Assistant Professor (80381268)
NOBUOKA Takayuki Sapporo Medical University, 1st Department of Surgery, Instructor (70359991)
|Project Period (FY)
2001 – 2003
Completed (Fiscal Year 2003)
|Budget Amount *help
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 2003: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2001: ¥6,100,000 (Direct Cost: ¥6,100,000)
|Keywords||hepatic failure / hepatosyte / stem cell / small hepatocyte / transplantation / cholestasis / bile canalicutus / sepsis / 重症感染症 / 非閉塞性胆汁うっ滞 / Cooplet細胞 / トランスポーター / ヒト肝細胞 / 遺伝子導入 / 細胞移植 / IPF-1 / アデノウイルスベクター / インスリン / 放射線照射 / 肝再生置換 / ミニ肝組織|
[Background] It is envisaged that hepatocyte transplantation and extracorporeal liver support systems may be a future alternative therapy to orthotopic liver transplantation. Small hepatocytes known as liver hepatocyte stem cells would be considered to be more adequate in hepatoyte transplantation. In addition, if a kind of specific function by a gene transfection to small hepatocytes was inducted, some benefits would provide for the patients with hepatic failure.
[Object] At first, small hepatocytes were determined as the objective cell source in hepatocyte transplantation. Whether alternative changes of contructed hepatocytes from recipient hepatocytes to small hepatocytes might occur or not has been studied. Therefore, PDX 1 gene, an insulin-producing gene, was tranfected to small hepatocytes by adenovial infection and those stem cells were analysed about the functional maintenance in vitro and in vivo. And the possibility of hepatosyte transplantation by those cells has been tried.
Experimental Classification] This research is composed of three kinds of experiments as shown as followings.
(1) Establishment of the transfection method of insulin-production gene. The transfection of PDX-1 gene, as an insulin-producing gene to adult hepatocytes and to small hepatocytes had been studied by the infection of adenoviral vectors. The expression of transfere genes, the production of pancreatic endocrine hormones and the characteristics of the hepatocytes were immurohis topathologically made sure in the transfected cells.
(2) Expression and production of insulin in the transfected cells. Immunohistologic determination of the transfected cells, analysis of m-RNA specific to the proteins, maintenance of function in the transfected cells were studied.
(3) Hepatocyte transplantation in vivo. Small hepatocyte or adult hepatocytes with or without transfection of the gene were transplanted into liver via the injection into spleen.
[Results] All of above research plans have been performed during 2001 to 2004, as shown in followings.
(1) Expression of several transfere factors were determined in the infected cells after the transfection of PDX-1. The quantitations of insulin, glucagon, somatostatin and pancreatic polypeptide in the cultured solution were succeeded. But it was very difficult to get the stable transfection and to repeat this experiment successfully.
(2) The transfection rates were extremely low but was observed higher in the adult hepatocytes than in small hepatocytes. The limitation of the maintenance of the function gained by transfection was strongly impressed on this research.
(3) Highly succeeded repopulation in the recipient liver was observed in the experiment with non gene transfected small hepatocytes and the proliferation of small hepatocyte in injected was also confident.
[Conclusion] This pilot research suggest that small hepatocytes transplantations would invite an entrance of the actuarial utilization of clinics. Less