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张顶, 高丽, 张远旭, 孙 丽, 冯 悦, 何有文, 夏雪山, 张华堂. 2009: 树鼩肝细胞体外分离培养体系的复建及主要影响因素分析. 动物学研究, 30(1): 24-30. DOI: 10.3724/SP.J.1141.2009.01024
引用本文: 张顶, 高丽, 张远旭, 孙 丽, 冯 悦, 何有文, 夏雪山, 张华堂. 2009: 树鼩肝细胞体外分离培养体系的复建及主要影响因素分析. 动物学研究, 30(1): 24-30. DOI: 10.3724/SP.J.1141.2009.01024
ZHANG Ding, GAO Li, ZHANG Yuan-xu, SUN Li, FENG Yue, HE You-wen, XIA Xue-shan, ZHAN. 2009: Crucial Factors for de novo Establishment of Long-term Primary Culture of Tree Shrew Hepatocytes. Zoological Research, 30(1): 24-30. DOI: 10.3724/SP.J.1141.2009.01024
Citation: ZHANG Ding, GAO Li, ZHANG Yuan-xu, SUN Li, FENG Yue, HE You-wen, XIA Xue-shan, ZHAN. 2009: Crucial Factors for de novo Establishment of Long-term Primary Culture of Tree Shrew Hepatocytes. Zoological Research, 30(1): 24-30. DOI: 10.3724/SP.J.1141.2009.01024

树鼩肝细胞体外分离培养体系的复建及主要影响因素分析

Crucial Factors for de novo Establishment of Long-term Primary Culture of Tree Shrew Hepatocytes

  • 摘要: 树鼩(Tupaia Belangeri)与人和灵长类亲缘关系较为接近,是乙型肝炎研究中备受关注的小动物模型,而其原代肝细胞的分离和培养则是建立HBV体外感染模型及应用和基础研究的关键的第一步,但由于以往的文献报道均较为简要,需要较长时间的摸索。本研究通过与机械分离法的直接比较,验证了两步灌注法在树鼩肝细胞分离中的优越性。进而发现,在分离后的体外培养过程中,二甲基亚砜不仅能够促进和维持原代肝细胞的分化,而且能够显著地抑制纤维状细胞群的出现。同时,肝细胞生长因子(HGF)和表皮生长因子(EGF)能够促进肝细胞在体外长期存活。在此优化的条件下,原代培养可持续4—5周,并且较多的细胞聚集形成类似肝窦结构的形态,从而为乙型肝炎病毒感染机理研究和药物筛选提供了必备的先决条件,也为丙型肝炎病毒和丁型肝炎病毒及单纯疱疹病毒等研究和药物筛选提供了可能。

     

    Abstract: Tree shrews(Tupaia Belangeri)are phenogenetically close to humans and primates and generally recognized as a prominent small animal model for studies of heptitis virus type B (HBV). Isolating and culturing tree shrew hepatocytes is the first key step toward the cellular model of HBV infection in vitro. Due to the lack of details in previous reports, establishing the model has been a matter of arbitary experiences. In this study, we validated the superiority of perfusion over mechanic dispersion for hepatocyte separation and isolation. Subsequent cultures showed that dimethyl sulphoxide (DMSO) could suppress the growth of fibroblast-like cells and maintain the hepatocytes in the differentiated status. Furthermore, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) could sustain growth and survival of the hepatocytes in the long-term culture. The combination of DMSO and HGF/EGF could maintain the hepatocytes in a longer and more stable differentiated status with clear trend to form liver sinus-like structures. Following this detailed method as a laboratory routine would permit plentiful starting material for study of HBV infection in vitro and drug-screening, as well as studies on hepatitis viruses type C and D, and herpes simplex virus.

     

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