A study in development of transgenic cattles for the use of hybrid bioartifical liver.

开发用于混合生物人工肝的转基因牛的研究。

• 批准号: 12309003
• 负责人: MAKUUCHI Masatoshi
• 金额: $ 30.31万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12309003/
• 关键词: Bioartificial liver; Transgenic animal; Transgenic mouse; Transgenic pig; human albumin DNA; EGFP; EGFP遺伝子; トランスジェニックマウス; トランスジェニックブタ; ハイブリッド型人工肝臓; トランスジェニック; マイクロマニピュレーション

In research on extracorporeal bioartificial liver perfusion system using xenogeneic liver, the chief purpose of the transgenic technique is to knock out genes of xenogeneic proteins, and transduce genes of human proteins into the animal. As an initial approach, we planned to transduce human albumin cDNA into porcine germ cells to produce porcine liver which can secret human albumin for the purpose of utilizing it for bioartificial liver support, we produced a gene of human albumin cDNA (hAlb) and EGFP (enhanced green fluorescent protein) gene, pCX-hAlb-EGFP. We transfected it initially into NIH3T3 cells using lipofection method, secondly into mouse germ cells by classic microinjection method, and thirdly into porcine germ cells by intracytoplasmic sperm injection (ICSI) method in order to induce expression of human albumin in the cultured cells and animal liver. As the results, NIH3T3 cells showed ultraviolet lightening and expressed human albumin in Northern hybridization analysis, and a transgenic pig was produced of which the skin showed ultraviolet lightening and integration of human albumin gene in PCR analysis. It was a pity that the pig was born dead, but this was initial epoch-making report of expression of EGFP and human albumin in TG pig produced by ICSI method.

在异种肝脏体外生物人工肝灌流系统的研究中，转基因技术的主要目的是敲除异种蛋白基因，并将人类蛋白基因导入动物体内。作为一种初步的方法，我们计划将人白蛋白基因转导到猪生殖细胞中，生产能够分泌人白蛋白的猪肝。为了将其用于生物人工肝支持，我们制备了人白蛋白基因(Halb)和增强型绿色荧光蛋白(EGFP)基因，pCX-halb-EGFP。我们首先用脂质体转染法将其导入NIH3T3细胞，然后用经典的显微注射法将其导入小鼠生殖细胞，最后用卵胞浆内单精子注射(ICSI)法将其导入猪生殖细胞，以诱导培养细胞和动物肝脏中人白蛋白的表达。Northern杂交结果表明，NIH3T3细胞有紫外线照射并表达人白蛋白，经PCR检测，获得了皮肤有紫外线照射并整合了人白蛋白基因的转基因猪。遗憾的是猪出生时死亡，但这是首次用ICSI方法在转基因猪中表达绿色荧光蛋白和人白蛋白的划时代报告。

项目成果

成瀬勝俊, 酒井康行: "再生医学における肝細胞の分離・再構築法"Surgery Frontier. 8. 69-75 (2001)

Katsutoshi Naruse，Yasuyuki Sakai：“再生医学中肝细胞的分离和重建方法”Surgery Frontier. 8. 69-75 (2001)。

Katutoshi Naruse: "Efficacy of non woven fabric bioreactor immobilized with porcine hepatoeyres for ex vivo xenogeneic perfusion treatment of liver failure in clogs"Artificial Organs. 25. 273-280 (2001)

Katutoshi Naruse：“固定有猪肝细胞的无纺布生物反应器用于体外异种灌注治疗木屐肝衰竭的功效”人工器官。

Katsuroshi Naruse: "Ellicary of non woven labre bioreactor immobilized with poreine hepalocyte for ex vivo xenogeneic perfusion treatment of liver failure in dogs."Artificial Organs. (in press). (2001)

Katsuroshi Naruse：“固定有毛孔肝细胞的无纺布生物反应器的椭圆体，用于离体异种灌注治疗狗的肝衰竭。”人工器官。

Katsutoshi Naruse, Masatoshi Makuuchi: "Development and perspectives of bioartificial liver support"Hepato-gastroenterology. 49. 91 (2002)

Katsutoshi Naruse、Masatoshi Makuuchi：“生物人工肝支持的发展和前景”肝胃肠病学。

Katsutoshi Naruse: "Development of xenogeneic direct hemoperfusion method in a bioartificial liver system"Journal of Artificial Organs. 5. 257-264 (2002)

Katsutoshi Naruse：“生物人工肝系统中异种直接血液灌流方法的开发”人工器官杂志。