中文摘要：

目的建立一种经济、稳定可靠的HSC分离方法,为体外研究提供细胞模型.方法 Hanks液在体灌洗大鼠肝脏,离体后用Ⅳ胶原酶、链蛋白酶、DNaseI消化肝脏,12%Nycodenz连续梯度液分离大鼠HSC,计数细胞得率,0.2%台盼蓝染色计算细胞活率.自发荧光、Desmin、α-SMA免疫荧光染色对HSC进行鉴定和纯度分析.结果分离HSC得率（3.7±0.6）×107/只大鼠,细胞活率〉90%,分离第1天自发荧光和第3天desmin染色阳性细胞〉90%.HSC随着体外培养形态明显改变,分离培养7 d后α-SMA阳性细胞〉90%,培养14 d或传代后〉95%.结论肝脏离体后消化能达到在体消化同样的效果,应用Nycodenz密度分离介质可获得满意的HSC得率和纯度.

英文摘要：

Objective To build an efficient procedure for isolating rat hepatic stellate cells. Methods Primary hepatic stellate cells （HSC） were isolated from normal Sprague-Dawley （SD） rats by infusion and combined digestion of pronase E and collagenase IV ex situ. Hepatic stellate cells were purified by density centrifugation with 12% Nycodenz. Autofluorescenes, desmin and smooth muscle actin （ a-SMA） immunofluorescence staining identified and assayed purity of HSC. HSC were activated by culture on uncoated plastic tissue culture dish and culture in a higher glucose Dulbecco＇s modified eagles medium （DMEM） supplemented with 10% fetal calf serum under 37~C contained 5% CO2 95% air incubater. Results The harvest rate of hepatic stellale cells was about （3.7 ＋ 0.6） x 107 per rat, and the viability was more than 90%. Hepatic stellate cells could be activated by culture for more than 7 days. Conclusion This reformed method is more efficient to isolate hepatic stellate cell and by culture the hepatic stellate cells can be activated.