ISOLATION AND CULTURE OF HEPATIC LIPOCYTES, KUPFFER CELLS, AND SINUSOIDAL ENDOTHELIAL-CELLS BY DENSITY GRADIENT CENTRIFUGATION WITH STRACTAN
ISOLATION AND CULTURE OF HEPATIC LIPOCYTES, KUPFFER CELLS, AND SINUSOIDAL ENDOTHELIAL-CELLS BY DENSITY GRADIENT CENTRIFUGATION WITH STRACTAN
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DOI：
10.1016/0003-2697(87)90673-7
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发表时间：
1987-02-15
影响因子：
2.9
通讯作者：
ROLL, FJ
中科院分区：
文献类型：
--
作者：
FRIEDMAN, SL;ROLL, FJ
描述了一种分离纯化肝脂肪细胞、库普弗细胞和适合培养的正弦内皮细胞的方法，使用密度梯度离心对多糖材料Stractan进行了描述。将正常大鼠或适当剂量维生素A处理的大鼠肝脏的非实质细胞消化液在6、8、12和20%层状梯度上分层：脂细胞与其他非实质细胞有效分离，并从梯度的顶部去除。Kupffer细胞和正弦内皮细胞在梯度中迁移到更密集的界面，分别通过差异镀和选择性胰蛋白酶化进一步纯化。在原代培养中，分离的无污染物的高活脂细胞粘附并逐渐扩散数天，并显示固有维生素A荧光和desmin阳性免疫染色。脂肪细胞在普通塑料上存活较长时间，这些细胞的胶原合成至少在28天内保持相对稳定。根据DNA含量的连续测定，原代培养的脂肪细胞在镀后7天开始增殖。用stracan密度离心分离的Kupffer细胞和窦状内皮细胞同样在培养中保持其典型的形态和功能特征；这些细胞分离物的纯度已通过特定的荧光标记得到证实。本研究表明Stractan密度梯度离心分离法是一种高效、灵敏、可重复的分离肝脏非实质细胞纯群体的方法。
A method for isolating purified populations of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells suitable for culture, using density gradient centrifugation on the polysaccharide material Stractan is described. A nonparenchymal cell digest of liver from either normal rats or rats treated with modest doses of vitamin A is layered on a discontinuous gradient of 6, 8, 12, and 20% Stractan: lipocytes are separated efficiently from other nonparenchymal cells and are removed from the top of the gradient. Kupffer cells and sinusoidal endothelial cells, which migrate to denser interfaces in the gradient, are further purified by differential plating and selective trypsinization, respectively. Isolated highly viable lipocytes free of contaminants adhere and spread progressively over several days in primary culture and display both intrinsic vitamin A fluorescence and positive immunostaining for desmin. Lipocytes survive for prolonged periods on plain plastic, and collagen synthesis by these cells remains relatively constant for at least 28 days. Based on serial assay of DNA content, lipocytes in primary culture proliferate, beginning 7 days after plating. Kupffer cells and sinusoidal endothelial cells isolated by Stractan density centrifugation likewise retain their typical morphologic and functional characteristics in culture; the purity of these cell isolates has been confirmed by using specific fluorescent markers. This investigation demonstrates that Stractan density gradient centrifugation is an efficient, sensitive, and reproducible method for isolating pure populations of hepatic nonparenchymal cells.