改良法大鼠肝星状细胞的分离培养及鉴定

An improved method of isolating and identifying rat hepatic stellate cells

目的 :改良大鼠肝星状细胞 (hepaticstellatecells,HSC)的分离培养及鉴定方法 ,使之简便易行 ,高效可靠。方法 :采用肝离体胶原酶灌注消化 ,低速离心去除肝细胞 ,密度梯度离心法分离HSC ,采用台盼蓝染色排斥法鉴定细胞活率 ,desmin加GFAP免疫细胞化学双染色法鉴定HSC纯度。结果 :采用改良法 ,每只大鼠的HSC得率为( 2 .5 4± 0 .13)× 10 7个 ,细胞活率为 ( 98.8± 0 .7) % ,高于旧方法的HSC得率 [( 2 .18± 0 .18)× 10 7个 ,P <0 .0 1]和活率 ( 94.3± 0 .6 ) % ,P <0 .0 1]。用desmin加GFAP免疫细胞化学双染色法鉴定 ,HSC纯度为 ( 96 .8± 1.0 ) % ,高于单用desmin鉴定的纯度 ( 91.8± 2 .2 ) % (P <0 .0 1)。结论 :改良的大鼠HSC分离培养方法简便易行又经济 ,在保证纯度的同时 ,提高了得率和活率。采用desmin加GFAP免疫细胞化学双染色法鉴定细胞纯度 。

Objective: To improve the method of isolating, culturing and identifying rat hepatic stellate cells(HSC). Methods: The liver was isolated and perfused with collagenase to obtain liver cell suspension. Then liver cell suspension was centrifuged at 50× g to remove hepatocytes. Lastly HSC were separated from other nonparenchymal cells by density gradient centrifugation. The cell viability was determined by Trypan blue exclusion staining. The purity of HSC was identified by the expression of either desmin or GFAP(glial fibrillary acidic proteins) using immunocytochemistry SP method. Results:The yield rate of HSC was (2.54±0.13)×10 7 per rat by modified method, which was higher than that [(2.18±0.18)×10 7 ]( P <0.01)of the former method. The cell viability was (98.8± 0.7)% by the modified method, which was higher than that [(94.3±0.6)%]( P <0.01) of the former method. The purity of HSC [(96.8±1.0)%] identified by double staining with labeled antibodies against desmin and GFAP was higher than that [(91.8±2.2)% ] ( P <0.01) of staining only with labeled antibody against desmin. Conclusion: The method of isolating and culturing HSC modified is simple, convenient, efficient and reliable. The method used to identify the purity of HSC is sensitive.