小鼠肝星状细胞的分离、培养及鉴定方法

Isolation,cultivation and identification of the BALB/c mice hepatic stellate cells

目的:探索BALB/c小鼠肝星状细胞(HSCs)分离、纯化的可行方案,并评价依该法分离所得HSCs的生物学特性。方法:经肝门静脉先用不含钙镁离子的Hank液充分灌洗,再以浓度为1mg/mL的Ⅳ型胶原酶灌注BALB/c小鼠肝脏。取肝后,完整分离后碾碎,37℃水浴振荡30min,Percoll连续密度梯度(60%)离心法分离、纯化HSCs,苔盼蓝染色检测HSCs活性,结蛋白免疫细胞化学鉴定HSCs,光镜观察体外培养HSCs的形态学变化。结果:纯化后每只小鼠HSCs收获量约为(5.5±0.4)×105个,HSCs纯度>90%,HSCs细胞活率>90%。结论:本实验建立的的分离纯化方案可获得高纯度高活率的小鼠HSCs。

Objective To establish a practical scheme for the isolation and purification of the hepatic stellate cells （HSCs） from BALB/c mice. Methods The HSCs were isolated from the liver of the BALB/c mice by Type IV collagenase （1 mg/ml） digestion and continuous density gradient centrifugation by 60% Percoll Solution. The viability of the isolated cells was determined by trypan blue staining assay. The HSCs were identified by immunocytochemical staining of Desmin. The morphologic changes of the HSCs were observed undera microscope. Results The average number of the HSCs from a single mouse liver was （5.5±0.4）×10^5, with purity and viability both over 90%. Conclusions This study established a practical scheme for the isolation and purification of the HSCs from the BALB/c mice.