目的 探索SD大鼠肝星状细胞(HSCs)的提取方法,观察其生物学特性,为进一步研究它在肝纤维化和免疫应答中的可能机制奠定基础.方法 依次采用前灌注液、0.15 mg/ml的链霉蛋白酶E和0.5mg/ml的Ⅳ型胶原酶在体原位灌注消化肝脏.肝脏离体剪碎后,在0.3mg/ml链霉蛋白酶E、0.3 mg/mlⅣ型胶原酶、0.02mg/ml DNA酶Ⅰ液中37℃振荡消化20min,低速离心(30g,5min)去除残余肝实质细胞,Optiprep密度梯度离心法获得纯化的HSCs.台盼蓝排斥试验评估HSCs细胞存活率；328nm紫外光激发HSCs自发蓝绿色荧光结合光镜鉴定细胞纯度；α-SMA免疫细胞化学方法鉴定HSCs;通过光镜观察HSCs的形态学变化.结果 本方法,每只大鼠肝脏HSCs产量约8.2×106个,HSCs存活率在90％以上；原代培养24h后,纯度在85％以上；传1代后,纯度达95％以上.结论 本方法简单、经济、实用,可获得较多的大鼠HSCs.%Objective To establish a practical scheme for the extraction of the hepatic stellate cells (HSCs) from Sprague Dawley rat, and observe their biological phenotypes, which could lay the foundation for the further study of the possible mechanism of it in liver fi-brosis and immune response. Methods The liver was digested by in situ perfusion of former perfusion solution, 0. 15mg/ml pronage E and 0. 5mg/ml collagenase Ⅳ in turn. The liver was continued to be cut into pieces and digest using 0. 3mg/ml pronage E, 0. 3mg/ml col-lagenase Ⅳ and 0. 02mg/ml DNage I solution with constant temperature oscillator for 20 min in vitro. After low speed centrifugation being used to remove residual hepatocytes, cells were treated with density gradient centrifugation by Optiprep to obtain the purified rat HSCs. Trypan blue exclusion test was used to estimate cell viability. The cell purity was identified by HSCs autofluorescence and light microscopy. The HSCs were identified by immunocytochemical staining of α - SMA. HSCs morphological changes were observed by light microscopy. Results This protocol yielded a preparation of approx 8. 2 × 10 cells from a single rat liver, with viability over 90% . The purity quotient of HSCs was more than 85% after 24 h of primary eluture and close to 95% after the first cell passage. Conclusion This method for isolating HSCs is simple, ecnomic and practical, which obtained more rat HSCs.
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