Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies
Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies
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DOI：
10.1002/pmic.201100157
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发表时间：
2011-09-01
期刊：
影响因子：
3.4
通讯作者：
Jiang, Ying
中科院分区：
文献类型：
--
作者：
Liu, Wei;Hou, Yufang;Jiang, Ying
越来越清楚的是，纯细胞群的分离为生物系统中的蛋白质谱分析提供了一种独特敏感和准确的方法，并为蛋白质组学分析开辟了新的领域。该方法首次将胶原酶密度梯度离心与磁活化细胞分选相结合，实现了高纯度、高产量的肝细胞、肝星状细胞、枯否细胞和肝窦内皮细胞的分离。超过98%的分离的HC是阳性的细胞角蛋白18，与91%的活力。约97%的分离的HSC表达胶质细胞酸性蛋白，存活率为95%。近98%的分离的KC表达F4/80，存活率为94%。LSECs纯度达91%，活力达94%。HC、HSC、LSEC和KCs的产量分别为每只小鼠6.3、1.3、2.6和5.0百万。这种系统的分离方法使我们能够以高纯度和高产量研究不同类型肝细胞的蛋白质组谱，这对于人类肝脏蛋白质组计划的样品制备特别有用。
It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.