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KMID : 0578319960060050534
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Molecules and Cells
1996 Volume.6 No. 5 p.534 ~ p.540
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Establishment of continuous Human Fetal hepatocyte Cell Line by Transfection of Simian Virus 40 T Gene
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Park, Joonwon
Lee, Hyo-Suk/Park, Joo Bae/Kim, chung Yong
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Abstract
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The problems of short-term survival and poor functional stability of human hepatocytes in culture has hindered investigators in using them as an in vitro system. Thus, the present study was designed to obtain an immortalized and differentiated human hepatocyte cell line by transfecting the cells with pSV3neo vector containing a simian virus 40 tumor (SV 40 T) gene and a neomycin selectable marker. A primary culture of human fetal hepatocytes obtained by therapeutic abortion at 18 weeks of gestation and grown in a serumfree chemically defined medium. SV40 T gene was transfected into the cells by using the polybren-DMSO method. Selected and transformed hepatocytes were subcultured in a 10% FBS supplemented medium. Morphological characteristics of subcultured cells were continually examined by phase-contrast microscope and electron microscope. The transfection of SV40 T gene was confirmed by immunofluorescence study using anti-SV40 T antigen after 72 h of subculture, and the differentiation of subcultured hepatocytes was proved by immunocytochemistry study using anti-human albumin and anti-human alpha fetoprotein. The cells were continuously replicating and maintained their morphological characteristics of epithelial origin for 30 subculturings over eight months. All the SV40 transformed hepatocyte cells stained positive for SV40 T antigen and were found to be positive for alpha fetoprotein and albumin, indicating differentiated hepatocyte lineage. These data suggested that we obtained a continuous human fetal hepatocyte cell line that secretes liver specific proteins.
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