非灌流法分离大鼠肝星形细胞及其鉴定

目的采用非灌流法分离大鼠肝星形细胞(Hepatic stellate cell,HSC),并进行鉴定。方法采用非灌流法结合酶消化法分离大鼠肝脏细胞,密度梯度离心进一步分离HSC,油红染色检测HSC胞质中的脂滴,免疫组化法检测细胞中α-平滑肌肌动蛋白(α-Smooth muscle actin,α-SMA)、结蛋白(Desmin)及神经胶质酸性蛋白(Glial fibrillary acidic protein,GFAP)的表达。结果非灌流法结合酶消化法可成功分离大鼠HSC;密度梯度离心纯化的HSC经油红染色,细胞核周围可见红色脂滴;该细胞中α-SMA、结蛋白及GFAP的免疫组化染色结果均呈阳性,细胞着色率可达90%以上。结论成功建立了大鼠HSC的非灌流分离模式,所获得的HSC纯度较高,该方法稳定简便,具有一定的推广应用价值。

Isolation of rat hepatic stellate cells by nonperfusion method and identification of isolated cells

Objective To isolate rat hepatic stellate cells(HSCs) by nonperfusion method and identify the isolated cells.Methods Rat liver cells were isolated by nonperfusion method combined with enzyme digestion method,from which HSCs were further isolated by density gradient centrifugation.The lipid droplets in cytoplasm of HSCs were determined by oil red staining,while the expressions of α-smooth muscle actin(α-SMA),desmin and neuroglia acid protein(GFAP) by immunohistochemical staining.Results Rat HSCs were successfully isolated by nonperfusion method combined with enzyme digestion method.After oil red staining,red lipid droplets were found around the nucleus in HSCs purified by density gradient centrifugation.All the positive rates of α-SMA,desmin and GFAP were more than 90% in immunohistochemical staining.Conclusion The nonperfusion isolating mode of rat HSC was established successfully,by which highly purified HSCs were obtained.The method was stable,simple,and worthy of popularization.