目的 改进大鼠肝星状细胞(HSC)的分离、纯化方法,为肝相关疾病研究提供稳定细胞来源.方法 采用严格控制灌注液温度、流速及灌注时间的肝逆向两步酶灌注法进行原位组织消化,并使用Pronase E/CollagenaseⅣ联合消化液体外消化肝组织,进而用Percoll密度梯度离心分离HSC细胞;台盼蓝拒染实验检测细胞存活率,CCK-8法测定细胞活力并制作细胞生长曲线;分别采用流式细胞术及免疫荧光细胞化学法对HSC细胞进行鉴定.结果 采用改良方法后,每只大鼠分离收获的HSC细胞稳定在(2.1±0.2)×10 7个,存活率为(96.2±0.8)%,细胞生长状态良好;流式细胞分选测定具有自发荧光特性的HSC细胞所占比例为96.3%,免疫荧光细胞化学法检测分离的HSC细胞表达特征性表面抗原α-SMA和Desmin.结论 采用严格控温、控流、控时的灌注方法及使用Pronase E/CollagenaseⅣ联合消化液有效提高了大鼠HSC细胞的收获数量、细胞活力和纯度,为进一步围绕HSC开展相关研究奠定了细胞基础.
Objeetive To improve the method for the isolation and purification of rat hepatic stellate(HSC) cells and to provide a stable cell source for the research on liver-related diseases.Methods Rat liver was digested in situ by a two-step infusion assay under a strict control of the infusion temperature,flow rate and time with a combined utilization of Pronase E and Collagenase Ⅳ.And then,the HSC cells were separated by Percoll density gradient centrifugation.The cell growth curve and survival rate were measured by CCK-8 and trypan blue staining,respectively.The HSC cells were identified by flow cytometry and immunofluorescence cytochemistry.Results With the improved methods,there were (2.1 ± 0.2) × 107 HSC cells isolated from one rat and the survival rate was (96.2 ± 0.8) %.The percentage of HSC cells with a spontaneous fluorescent characteristic from the isolated cells was 96.3%.The immunofluorescence cytochemistry was used to detect the expressions of the surface antigens α-SMA and Desmin in the isolated HSC cells.Conclusion By strict control of infusion temperature,flow rate and perfusion time as well as the combined application of Pronase E and Collagenase Ⅳ,there is an increased harvest of HSC cells with improved cell viability and purity,which is helpful for further research on HSC cells.