基于Cre/loxP的人肝细胞可逆性永生化 1.

Cre/loxP-Based Reversible Immortalization of Human Hepatocytes 1.

作者信息

Kobayashi Naoya, Noguchi Hirofumi, Westerman Karen A, Watanabe Takamasa, Matsumura Toshihisa, Totsugawa Toshinori, Fujiwara Toshiyoshi, Leboulch Philippe, Tanaka Noriaki

机构信息

First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.

Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA 02139.

出版信息

Cell Transplant. 2001 May/Jul;10(4-5):383-386. doi: 10.3727/000000001783986558.

DOI:10.3727/000000001783986558

PMID:28886302

Abstract

An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.

摘要

用于肝细胞移植的理想替代原代人肝细胞的方法是使用一种克隆细胞系，该细胞系在培养中能经济地生长，并且在移植后表现出分化的、未转化的肝细胞的特征。本研究的目的是建立一种可逆永生化的人肝细胞系。用人源化逆转录病毒载体SSR#69使人类肝细胞永生化，该载体表达由一对loxP重组靶点侧翼的猿猴病毒40大T抗原（SV40Tag）基因。所得到的一个克隆NKNT-3表现出肝实质细胞的形态特征，并表达分化肝功能的基因。NKNT-3细胞具有无限可用性。在腺病毒递送Cre重组酶并随后进行差异选择后，从NKNT-3细胞中有效去除了SV40Tag。在此我们表明，逆转录病毒转移的SV40Tag基因的消除可通过腺病毒介导的位点特异性重组切除。