Construction of hybrid liver support system using gene amplified recombinant mammalian cells

使用基因扩增重组哺乳动物细胞构建混合肝脏支持系统

• 批准号: 07558125
• 负责人: SUGA Ken-ichi
• 金额: $ 8.06万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558125/
• 关键词: hybrid artificial liver support system; recombinant DNA; ammonia removal; drug metabolism; gene amplification; glutamine synthetase; 人工肝臓; 組伝子組換え; 人工肝; MSX; GS; アンモニア

In order to establish the cell line which was suitable for hybrid artificial liver support system, an ammonia metabolizing activity was given to Chinese Hamster Ovary (CHO) and HepG2 cell line using genetic engineering techniques. CHO-K1 and HepG2 cells were transformed with pSV2-GS vector which contains glutamine synthetase gene connected with SV40 or CMV promoter. The recombinant cell line was selected under the various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The obtained MSX tolerable cell line had the high glutamine synthetase activity and could remove the ammonium from medium. The kinetic parameters of this cell line were determined under 0,1, or 2mM ammonia concentration. The specific ammonium consumption rates of established CHO and HepG2 cell line during growth phase were about one fourth and one seventh of that of primary hepatocyte respectively.

为建立适合于混合型人工肝支持系统的细胞系，利用基因工程技术对中国卵巢癌（CHO）细胞系和HepG 2细胞系进行了氨代谢活性的改造。将含有谷氨酰胺合成酶基因的pSV 2-GS载体与SV 40或CMV启动子连接，转化CHO-K1和HepG 2细胞。在不同浓度的谷氨酰胺合成酶抑制剂甲硫氨酸亚砜亚胺（MSX）下筛选重组细胞系。所获得的耐MSX细胞系具有较高的谷氨酰胺合成酶活性，并能去除培养基中的铵离子。在0、1和2 mM氨浓度下测定了该细胞系的动力学参数。已建立的CHO和HepG 2细胞系在生长期的比氨消耗率分别约为原代肝细胞的1/4和1/7。

项目成果

Takeshi Omasa, Naoko Tanimura, Shigeo Urayama, Mitsugu Yamanaka, Yoshio Katakura, Michimasa Kishimoto, and Ken-ichi Suga: "Construction and evaluation of ammonia metabolizing cell line for hybrid bioartificial liver support system using recombinant DNA te

Takeshi Omasa、Naoko Tanimura、Shigeo Urayama、Mitsugu Yamanaka、Yoshio Katakura、Michimasa Kishimoto 和 Ken-ichi Suga：“使用重组 DNA 技术构建和评估用于混合生物人工肝支持系统的氨代谢细胞系

Suga et al.: ""Construction and evaluation of ammonia metabolizing cell line for hybrid bioartificial liver support system using recombinant DNA techniques"" 化学工学シンポジウムシリーズ. 61. 187-191 (1998)

Suga 等人：“使用重组 DNA 技术构建和评估混合生物人工肝支持系统的氨代谢细胞系”，化学工程研讨会系列，61. 187-191 (1998)。

Suga et al.: ""Construction and evaluation of ammonia metabolizing cell line for hybrid bioartificial liver support system using recombinant DNA techniques"" In;"Biochemical Engineeering:Marching toward the Century of Biotechnology"Zhongyao Shen,Fan Ouyan

Suga等：“利用重组DNA技术构建和评估用于混合生物人工肝支持系统的氨代谢细胞系”，载于；“生化工程：迈向生物技术世纪”沉中耀，范欧燕

K.Suga, et al.: ""Construction and evaluation of ammonia metabolizing cell line for hybrid bioartificial liver support system using recombinant DNA techniques"" In;"Biochemical Engineering:Marching toward the Century of Biotechnology"Zhongyao Shen,Fan Ouy

K.Suga等人：“利用重组DNA技术构建和评估用于混合生物人工肝支持系统的氨代谢细胞系”，载于；“生物化学工程：迈向生物技术的世纪”Zhongyao Shen，Fan Ouy

Takeshi Omasa, Shigeo Urayama, Mitsugu Yamanaka, Naoko Tanimura, Yoshio Katakura, Michimasa Kishimoto, and Ken-ichi Suga: "Construction and evaluation of ammonia metabolizing cell line for hybrid bioartificial liver support system using recombinant DNA te

Takeshi Omasa、Shigeo Urayama、Mitsugu Yamanaka、Naoko Tanimura、Yoshio Katakura、Michimasa Kishimoto 和 Ken-ichi Suga：“使用重组 DNA 技术构建和评估用于混合生物人工肝支持系统的氨代谢细胞系