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目的建立和改良人肝脏星状细胞(human-hepatic stellate cells,hu-HSCs)分离方法,使之适于原代培养和研究。方法在传统分离HSCs方法的基础上,省略链霉蛋白酶和DNA酶,用EGTA液促细胞解离,再用Ⅳ型胶原酶消化,差速离心去除肝实质细胞,最后用Nycodenz密度梯度离心纯化hu-HSCs,台盼蓝染色法计算细胞产量和进行活力评估,α-SMA、desmin免疫荧光染色及油红O脂滴染色鉴定细胞纯度。结果分离得到的原代hu-HSCs数量高达3×106个/克肝脏,纯度达98%;细胞传代后纯度达到100%。结论改良后的方法经济、快捷、高效,细胞得率及纯度大为提高,有利于建立在原代hu-HSCs基础上的研究。
Objective To establish and improve the method of human hepatic stellate cells (hu-HSCs) isolation and to make it suitable for primary culture and research. Methods Based on the traditional method of HSCs isolation, pronase and DNase were omitted, and EGTA was used to dissociate the cells. The cells were digested with type Ⅳ collagenase and the liver parenchymal cells were removed by differential centrifugation. Finally, the cells were purified with Nycodenz density gradient centrifugation -HSCs and trypan blue staining were used to evaluate cell viability. Α-SMA, desmin immunofluorescence staining and oil red O lipid drop staining were used to identify cell purity. Results The number of primary hu-HSCs isolated was as high as 3 × 106 / g liver with a purity of 98%. The purity of the cells reached 100% after passage. CONCLUSION: The improved method is economical, rapid and efficient, and the cell yield and purity are greatly improved, which is beneficial to the research based on primary hu-HSCs.