Production of transgenic pigs for the use of bioartificial liver.

生产用于生物人工肝的转基因猪。

• 批准号: 15109008
• 负责人: MAKUUCHI Masatoshi
• 金额: $ 76.04万
• 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 (S)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15109008/
• 关键词: Transgenic pig; Albumin; EGFP; ヒト血清アルブミン; 人工肝臓; 人血清アルブミン; 動物バイオリアクター

Producing transgenic cattle that can secret human proteins is a promising strategy for development of a bioreactor for human pharmaceuticals or bioartificial liver support. We constructed pCX-hAlb-EGFP, that is, a combined gene of human albumin cDNA and EGFP cDNA connected with CMV-IE enhancer, chicken β-actin promoter, and rabbit β-globin poly A signal for the purpose of producing a transgenic pig that can secret human albumin systemically. NIH3T3 cells transformed with pCX-hAlb-EGFP by the lipofection method showed intense expression of human albumin and EGFP genes. Thus, we introduced it into porcine oocytes using sperm vector method, and a piglet was produced which showed clear expression of GFP in the hooves and skin. PCR analysis of genomic DNA extracted from several tissues including the liver showed that they possessed the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the same tissues. The transgene was integrated and expressed in the porcine liver, which is indispensable for our purpose of utilizing the transgenic liver for bioartificial liver support. Real time RT-PCR analysis exhibited that the expression rate of human albumin gene and EGFP gene against β-actin promoter in the liver showed adequate quantity of transcribed mRNA and fairly good expression rates as compared with those of the other tissues. These results will promise successful result of further trials of producing desirable transgenic pig with the liver secreting human albumin.

生产能够分泌人类蛋白质的转基因牛是开发用于人类药物或生物人工肝支持的生物反应器的有前途的策略。为了获得系统分泌人白蛋白的转基因猪，我们构建了pCX-hAlb-EGFP，即人白蛋白cDNA和EGFP cDNA的联合基因，并连接了CMV-IE增强子、鸡β-actin启动子和兔β-珠蛋白poly A信号。用脂质体法将pCX-hAlb-EGFP转化NIH 3 T3细胞后，人白蛋白和EGFP基因均得到高效表达。因此，我们使用精子载体法将其导入猪卵母细胞，并产生了在蹄和皮肤中显示出清晰表达GFP的仔猪。从包括肝脏在内的几种组织中提取的基因组DNA的PCR分析表明，它们具有转基因。RT-PCR分析表明，人白蛋白和EGFP基因在同一组织中表达。转基因在猪肝中的整合和表达，是我们利用转基因肝作为生物人工肝支持的必要条件。真实的时间RT-PCR分析显示，人白蛋白基因和针对β-actin启动子的EGFP基因在肝脏中的表达率与其他组织相比，显示出足够数量的转录mRNA和较好的表达率。这些结果将为进一步生产肝脏分泌人白蛋白的转基因猪的试验提供成功的结果。

项目成果

成瀬勝俊: "人工肝臓と医療用形質転換ブタの開発"先端医療シリーズ25 肝胆膵の最新医療. 83-92 (2003)

Katsutoshi Naruse：“人工肝脏和医学转化猪的开发”高级医学系列 25 肝脏、胆汁和胰腺的最新医学治疗 83-92 (2003)。

Naruse K, Sakai Y, Natori T, Guo L, Shindo J, Iida Y, Michishita K, Karasawa Y, Kojima K, Makuuchi M: "Xenogeneic direct hemoperfusion using whole swine liver for liver failure in dogs"Journal of Surgical Research. 111. 229-235 (2003)

Naruse K、Sakai Y、Natori T、Guo L、Shindo J、Iida Y、Michishita K、Karasawa Y、Kojima K、Makuuchi M：“使用整个猪肝进行异种直接血液灌流治疗狗的肝衰竭”外科研究杂志。

Development and perspectives of perfusion treatment for liver failure

肝衰竭灌注治疗的进展与展望

• 发表时间: 2005
• 期刊: Surgery Today 35(7)
• 作者: J. Arita;K. Hasegawa;N. Kokudo;K. Sano;Y. Sugawara and M. Makuuchi;Naruse K. Nagashima H. Sakai Y. Kokudo N. Makuuchi M.
• 通讯作者: Naruse K. Nagashima H. Sakai Y. Kokudo N. Makuuchi M.

Efficacy of xenogeneic direct hemoperfusion using whole swine liver for liver failure in dogs.

使用猪全肝进行异种直接血液灌流治疗犬肝衰竭的疗效。

• 发表时间: 2003
• 期刊: Journal of Surgical Research 111
• 作者: Naruse K.;Sakai Y.;Naroti T.;Guo L.;Shindoh J.;Michishita K.;Iida Y.;Makuuchi M.
• 通讯作者: Makuuchi M.

成瀬勝俊: "スタンダードとなり得る人工肝臓システムの開発"現代医療. 36巻別刷. 135-142 (2003)

Katsutoshi Naruse：“可成为标准的人工肝脏系统的开发”，《现代医学》第 36 卷重印（2003 年）。