上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (5): 561-.doi: 10.3969/j.issn.1674-8115.2011.05.008

• 论著（基础研究） • 上一篇    下一篇

大鼠肝脏卵圆细胞干细胞特性的研究

李 影, 赵丽娟, 李艳香, 施 超, 陆颖理   

  1. 上海交通大学 医学院附属第九人民医院内分泌代谢科, 上海 2000l1
  • 出版日期:2011-05-28 发布日期:2011-05-27
  • 作者简介:李 影（1984—）, 女, 硕士生；电子信箱: liying1631230@163.com。
  • 基金资助:

    上海交通大学医工(理)交叉研究重点基金(YG2009ZD204)

Characteristics of stem cells from hepatic oval cells in rats

LI Ying, ZHAO Li-juan, LI Yan_xiang, SHI Chao, LU Ying-li   

  1. Department of Endocrinology and Metabolism, the Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, |China
  • Online:2011-05-28 Published:2011-05-27
  • Supported by:

    Foundation from Shanghai Jiaotong University, YG2009ZD204

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摘要：

目的 探讨大鼠肝脏卵圆细胞体外培养扩增及其干细胞特征。方法 采用2-乙酰氨基芴/四氯化碳方法建模，胶原酶灌流和流式细胞术分选纯化卵圆细胞，进行培养和扩增。透射电子显微镜观察肝脏卵圆细胞的超微结构，免疫细胞化学法检测干细胞标志物OV-6、c-kit和Thy-1.1的表达；RT-PCR法检测卵圆细胞内c-kit、肝细胞标志物（HNF-4α、AFP）和胆管细胞标志物（CK-7、CK-19）的基因表达；Western blotting法检测OV-6和AFP蛋白的表达。结果 大鼠肝脏组织可见明显的卵圆细胞增生，成簇分布，逐渐向肝实质内延伸。细胞体外培养可达40 d以上，细胞体积较小，多呈圆形和卵圆形。透射电子显微镜下可见细胞直径5～10 μm，细胞核大，胞质少，核/质比例大，细胞器少，仅见少量线粒体和内质网。免疫细胞化学检测显示，细胞表面OV-6、c-kit和Thy-1.1染色阳性；RT-PCR检测显示，卵圆细胞内有c-kit、HNF-4α、AFP、CK-7和CK-19基因表达；Western blotting检测显示，OV-6蛋白持续表达，AFP蛋白表达逐渐增强。结论 大鼠肝脏卵圆细胞体外可大量扩增并且保留其干细胞和双向分化潜能特性。

关键词: 卵圆细胞, 增殖, 体外, 干细胞

Abstract:

Objective To investigate the in vitro culture and characteristics of stem cells of hepatic oval cells in rats. Methods Rats were treated with 2-acetylaminofluorene and carbon tetrachloride to establish the models of hepatic oval cells, two-step collagenase perfusion technique and flow cytometry sorting were used to separate and purify oval cells, and cell culture and amplification were performed. The ultrastructure of hepatic oval cells was observed by transmission electron microscopy, and the expression of stem cell markers OV-6, c-kit and Thy-1.1 were detected by immunocytochemistry. The expression of c-kit, hepatic cell marker (HNF-4α and AFP) and bile duct cell marker (CK-7 and CK-19) gene in oval cells was detected by RT-PCR. The expression of OV-6 and AFP protein was determined by Western blotting. Results Obvious oval cell proliferation was observed in rat liver tissues, distributing as cluster and gradually extending to liver parenchyma. These cells could be cultured in vitro up to 40 d, with small size and ovoid nuclei. It was observed under transmission electron microscopy that the cell diameters ranged from 5 μm to 10 μm, and there were high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. It was revealed by immunocytochemistry that there was positive staining of OV-6, c-kit and Thy-1.1 on cell surface. RT-PCR analysis indicated that there was expression of c-kit, HNF-4α, AFP, CK-7 and CK-19 gene in oval cells. Western blotting demonstrated that there was continuous expression of OV-6 protein, and the expression of AFP protein gradually increased. Conclusion Hepatic oval cells derived from rat livers possess a high potential to proliferate in vitro, while maintaining the bipotential nature of stem cells.

Key words: oval cells, proliferation, in vitro, stem cells