Primary isolated hepatic oval cells maintain progenitor cell phenotypes after two-year prolonged cultivation
Primary isolated hepatic oval cells maintain progenitor cell phenotypes after two-year prolonged cultivation
复制标题
原代分离的肝卵圆细胞经过两年的长期培养后仍保持祖细胞表型。
DOI：
10.1016/j.jhep.2010.05.014
复制
发表时间：
2010-11-01
影响因子：
25.7
通讯作者：
You, Hong
中科院分区：
文献类型：
--
作者：
Wang, Ping;Cong, Min;You, Hong
背景和目标：尽管可扩增的肝祖细胞为肝脏疾病的治疗提供了可再生的细胞来源，但肝祖细胞的长期培养可能影响增殖和分化能力，甚至引发恶性肿瘤干细胞的形成。本研究的目的是确定长期培养后的原代培养的肝卵圆细胞的特性在vitro.Methods：肝卵圆细胞分离与胆碱缺乏，乙硫氨酸补充的饮食喂养大鼠连续繁殖，每5-7天，100代超过两年。肝细胞分化诱导丁酸钠和其特征在于使用蛋白质印迹，高碘酸希夫测定，白蛋白分泌和尿素生产。采用生长曲线和细胞周期分析评价其增殖能力;采用软琼脂和异种移植试验测定其非贴壁生长和致瘤性。连续传代2年后，具有典型上皮形态的肝卵圆细胞持续表达OV-6、BD-1、BD-2和Dlk作为肝祖细胞的标记，细胞角蛋白19作为胆管细胞标记，以及甲胎蛋白和白蛋白作为肝细胞标志物。丁酸钠还能诱导这些细胞分化为具有白蛋白分泌和氨清除尿素生成功能的糖原储存细胞，提示其向肝细胞分化。肝卵圆细胞在第50代后增殖略有加快，但仍保持二倍体细胞，染色体稳定，不具备非锚定依赖性生长能力，在免疫缺陷小鼠体内不发生肿瘤，提示无自发性恶性转化。肝卵圆细胞在长期培养后保持祖细胞特征而不发生自发性恶性转化，因此可以作为将来开发干细胞技术的可扩增细胞来源。(C)2010年欧洲肝脏研究协会。Elsevier B. V.出版，保留所有权利。
Background & Aims: Although expandable hepatic progenitors provide renewable cell sources for treatment of hepatic disorders, long-term cultivation of hepatic progenitors may affect proliferation and differentiation abilities, and even initiate the formation of malignant cancer stem cells. This study aims to determine characteristics of primary cultured hepatic oval cells after prolonged cultivation in vitro.Methods: Hepatic oval cells isolated from rats fed with a choline-deficient, ethionine-supplemented diet were continuously propagated every 5-7 days, to 100 passages over two years. Hepatocytic differentiation was induced by sodium butyrate and characterized using western blot, periodic acid Schiff assays, albumin secretion and urea production. Proliferation capacity was, evaluated using growth-curve and cell-cycle analysis; anchorage-independent growth and tumorigenicity were determined using soft agar and xenograft assay.Results: After 2 years of serial passages, hepatic oval cells with typical epithelial morphology continuously expressed OV-6, BD-1, BD-2, and Dlk as markers for hepatic progenitors, cytokeratin 19 as a cholangiocyte marker, and alpha-fetoprotein and albumin as hepatocyte markers. Furthermore, sodium butyrate could induce these cells to become glycogen-storage cells with the functions of albumin secretion and ureagenesis from ammonia clearance, indicating hepatocytic differentiation. Although proliferation slightly accelerated after the 50th passage, hepatic oval cells stayed diploid cells with features of chromosomal stability, which did not acquire anchorage-independent growth capacity and caused no tumor in immunodeficient mice, suggesting no spontaneous malignant transformation.Conclusions: Hepatic oval cells retain the progenitor cell features without spontaneous malignant transformation after prolonged cultivation, and thus may serve as an expandable cell source for future exploitation of stem cell technology. (C) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.