大鼠肝星状细胞分离与培养方法的实验研究(1)
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[摘要] 目的:探讨一种经济易行的分离与培养大鼠肝星状细胞的方法,为研究肝纤维化的发病机制提供良好条件。方法:采用离体循环酶液灌注法对成年大鼠的肝脏进行消化,经两种密度的Nycodenze密度梯度液高速离心(20 000 r/min)和细胞悬液低速离心(40× g)去杂质后得到肝星状细胞。台盼蓝拒染实验鉴定细胞活率,desmin和GFAP免疫细胞化学双抗单染色法鉴定肝星状细胞的纯度。结果:肝星状细胞的得率为(3.6±0.1)×107/肝,肝星状细胞存活率为(92.8±0.8)%,对培养24 h的肝星状细胞行desmin和GFAP共同鉴定DAB显色,细胞阳性率为(96.2±0.5)%。结论:本改良法在保证大鼠肝星状细胞产量的前提下提高了细胞纯度,有利于对原代大鼠肝星状细胞及肝纤维化的进一步研究。
[关键词] 肝星状细胞;细胞分离;原代培养;细胞鉴定
[中图分类号] R329.2+8[文献标识码]A [文章编号]1673-7210(2010)05(b)-030-03
The experimental study of rat hepatic stellate cells separation and cultivation
SHI Huimin1, XU Junquan2, WANG Dengni2, WANG Mingliang2, SONG Binyu2
(1.Shanxi Medical University, Taiyuan 030001, China; 2.Shanxi Medical University Fenyang Collage, Fenyang 032200, China)
[Abstract] Objective: To explore an economical and easy method for isolating and culturing the rat hepatic stellate cells to study the pathogenesis of hepatic fibrosis. Methods: Adult healthy rats' isolated livers were perfused by cyclophorase liquid in vitro. After the density gradient centrifugation of nycodenze at the speed of 20 000 r/min and the low-speed centrifugation (40× g) of cell suspension, the higher purity hepatic stellate cells were obtained. The refusal rate of cells could identify the rate of live cells in the trypan blue test; desmin and GFAP immunocytochemistry (double antibodies and single staining) were used to identify the purity of hepatic stellate cells. Results: The yield of hepatic stellate cells was (3.6±0.1)×107 per liver, the survival rate of hepatic stellate cells was (92.8±0.8)%, HSCs cultured for 24 hours were identified by desmin and GFAP, single staining the rate of positive cell was (96.2±0.5)%. Conclusion: The improved method increases the purity of the primary rat hepatic stellate cells on the base of promising the yield in order to study the primary hepatic stellate cells and liver fibrosis.
[Key word] Hepatic satellite cells; Cell isolation; Primary culture; Cell identification
肝星状细胞(hepatic satellite cells,HSCs)是肝脏内的一种非实质细胞,占整个肝脏的8%～13%[1-2] ......
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