|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Purpose : The goal of this research is to establish the bioreactor using immortalized human hepatocytes as an artificial liver.
1) Construction of bioreactor ; The radialflow type bioreactor was constructed. The abilities of albumin production and anmonia metabolism were demonstrated.
2) Establishment of human immortalized hepatocytes ; The establishment of immortalized hepatocytes was essential because human hepatocytes they were supposed to be given could not be obtained with the problem of copyright. First, reversible immortalized hepatocytes in rats could be established using SSR#69 (Retrovirus containing SV-40, lox-P)and AxCACre. Human hepatocytes were isolated and cultured using liver resection material and to which the gene was transduced, however, the immortalization has been able to be succeeded so far. The gene transduced could not function and therfore largeT-antigen could not be recognized by western blotting.
3) Liver failure model in pigs ; The liver failure model was established after portacaval shunt and ligation of hepatic artery. All pigs died within 12 hours.
Establishment of immortalized hepatocytes in human is essential for thea goal of this study. One of the reasons why effective transduction could not be obtained is though to be ischemic change from the resection of liver to the cell culture. The arrangement of this procedure is needed. After the establishment of immortalized hepatocytes in human, we go ahead for the application to bioreactor.
不死化細胞の作製については,成人ヒト肝細胞に対し,良好な遺伝子導入がえられないことの原因として臓器摘出からコラゲナーゼ処理までの虚血障害が考えられるため,今後臓器摘出後の手技,方法を検討する必要があると考える。またヒト不死化肝細胞作製後はラジアルフロー型バイオリアクターを用いて,アルブミン,凝固因子の合成能,グルクロン酸抱合能,アンモニア代謝能,各種負荷テストを行い,in vitroで人工肝臓としての機能評価を行うとともに,ブタ急性肝不全モデルを用いたin vivoでの機能評価を行っていく予定である。