不同二步酶灌注法分离大鼠肝星状细胞的比较研究

目的:在现有二步酶灌注法分离大鼠肝星状细胞(hepatic stellate cells,HSC)的基础上,探索更加高效的分离HSC方法。方法:分别采用链酶蛋白酶+胶原酶循环灌注、链酶蛋白酶非循环灌注+胶原酶循环灌注以及胶原酶单独循环灌注法分离大鼠HSC,比较三种方法的细胞获得率、活性和纯度差异。应用0.4%台盼蓝染色判断活性,结蛋白(desmin)、波形蛋白(vimentin)细胞免疫荧光方法鉴定纯度。结果:链酶蛋白酶非循环灌注+胶原酶循环灌注法细胞获得率高于另两种方法,细胞活力高于链酶蛋白酶循环灌注+胶原酶循环灌注法,三组得到的细胞纯度均高于90%且无显著差异。结论:在三种二步酶灌注方法中,链酶蛋白酶非循环灌注+胶原酶循环灌注法能显著提高HSC获得率,且对细胞活力影响小,不降低细胞纯度,是一种高效的分离方法,有利于HSC相关肝脏疾病的生物学研究。

Comparative Study on Isolation of Rat Primary Hepatic Stellate Cells by Different Two-step Enzyme Perfusion

Objective:To explore a more efficient method for HSC isolation based on the method of the two-step enzymatic infusion.Methods:Three methods including circulatory pronase+circulatory collagenase perfusion, non-circulatory pronase + circulatory collagenase perfusion and single collagenase perfusion were performed, and cell yield rate, viability and purity were compared. Cells viability was examined by 0.4% trypan blue. Desmin and vimentin immunofluorescence was used to identify cell purity.Results:The yield of HSC by non-circulatory pronase + circulatory collagenase perfusion was higher than that of the other two methods, and cell viablity was higher than that by circulatory pronase + circulatory collagenase perfusion. Cell purity of all groups was over 90%, and there was no significant difference.Conclusion:Among the three two-step enzymatic perfusion methods, non-circulatory pronase + circulatory collagenase perfusion can significantly improve the obtaining rate of HSC, and have little effect on cell viability without reducing cell purity, which is a highly efficient isolation method and benefits the biological research for HSC related liver diseases.