| 高活力原代小鼠肝细胞的分离与纯化 |
| 投稿时间：2010-11-13 点此下载全文 |
| 引用本文：王燕,周露婷,章卫平,谢志芳.高活力原代小鼠肝细胞的分离与纯化[J].医学研究杂志,2011,40(3):28-31 |
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| 摘要点击次数: 1994 |
| 全文下载次数: 2008 |
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| 基金项目:国家自然科学基金资助项目（30772478，31025013） |
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| 中文摘要:目的建立一种稳定的能获得高活力和高纯度原代小鼠肝细胞的分离和纯化方法。方法对传统的两步原位胶原酶灌注法进行4个方面的优化，主要包括选择逆向灌流、将持续灌注法改为间断灌注后夹闭门静脉法、严格控制胶原酶消化时间和将分离的肝细胞悬液进行低速Percoll单密度离心纯化。分离后的肝细胞进行体外培养。肝细胞活力和得率用台盼蓝染色法检测。结果新鲜分离的小鼠原代肝细胞活力能稳定达到87%±3％，平均活细胞总数为8×105每克小鼠体重，绝大部分细胞在体外培养2h后贴壁生长。结论优化后的肝细胞分离方法更为稳定、有效，分离到的肝细胞具有高活力的优点。 |
| 中文关键词:小鼠肝细胞 分离 灌注 原代培养 |
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| Isolation of Primary Mouse Hepatocytes with High Viability |
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| Abstract:ObjectiveTo develop a consistent method for the isolation of primary mouse hepatocytes with high-viability. MethodsThe conventional two-step collagenase perfusion technique was modified in the following 4 steps, including retrograde perfusion, intermittent perfution followed by occlusion of portal vein, optimization of enzymatic digestion, and purification using a low-speed, iso-density percoll centrifugation. Following isolation, the cells were maintained in a culture medium. The cell viability and yield were determined by trypan blue exclusion. ResultsThe isolations using this method resulted in an average yield of 8×105 viable cells / gram body weight and the cell viability of 87%±3％. Most viable hepatocytes attached to plates within 2h. ConclusionThe improved method is more consistent and efficient for isolating primary mouse hepatocytes with high-viability. |
| keywords:Mouse hepatocytes Isolation Perfusion Primary culture |
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