目的 改良肝星状细胞(HSC)的分离方法,为深入研究肝纤维化的发生机制奠定基础.方法 采用Ⅳ型胶原酶和链酶蛋白酶原位消化肝脏,在1.040~1.060 g/mL范围内多重多次密度梯度离心分离HSC,台盼蓝染色测定细胞存活率,328 nm紫外光激发HSC自发蓝绿色荧光法和Desmin细胞免疫荧光法鉴定HSC及检测HSC纯度.Desmin和α-SMA细胞免疫荧光方法鉴定HSC静息和活化状态.结果 每只大鼠肝脏HSC得率为(3~5)×107个,HSC存活率在95%以上,纯度在90%以上,体外培养14 d后活化的HSC纯度几乎达100%.结论 在1.040~1.060 g/mL密度范围内采用多重多次密度梯度离心方法,避免了HSC分离过程中因密度配比误差及HSC自身密度相对不均一性所导致的细胞流失,建立了一种改良的肝星状细胞分离方法,HSC得率和纯度更加稳定,达到国外文献报道水平.%Objective To improve the method of isolating rat hepatic stellate cells (HSCs). Methods Rat hepatic stellate cells were isolated for several times with multiple density centrifugation within 1. 040 ～ 1. 060 g/mL after enzyme perfusion of liver. The cell viability was determined by Trypan blue exclution staining. The purity of HSCs was identified by retinoid antofluorescence and immunostaining by Desmin. The phenotype of HSCs was evaluated by characteristic immunofluorescence using antibodies specific for Desmin and α-SMA. Results About (3 ～5 ) × 107 HSCs were harvested from each rat through multiple density centrifugation within 1. 040 ～ 1.060 g/mL. The cell viability was more than 95％ determined by the trypan blue exclusion method. The purity of freshly isolated HSCs and the activated HSCs were more than 90％ and almost 100％ respectively assessed by retinoid antofluorescence and immunostaining cells using Desmin antibody. Conclusion The method of multiple density centrifugation greatly increased the production of HSCs by reducing loss of cells due to error of density preparation and their relative heterogeneity of density during isolating HSCs. This method is more convenient, efficient and reliable.
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