Optiprep法分离大鼠肝星状细胞的实验研究

Experimental study on isolation of hepatic stellate cell in rats by Optiprep

目的:建立一种合理、经济、便捷的大鼠原代肝星状细胞(hepatic stellate cells,HSC)分离方法。方法:运用改良肝脏原位灌注D-Hanks后,改用Ⅳ型胶原酶和DNA酶Ⅰ离体灌注,10%、16%Optiprep密度梯度离心分离大鼠肝星状细胞,台盼蓝排斥试验鉴定细胞活力,α-SMA免疫细胞化学法鉴定细胞纯度。结果:我们采用Optiprep密度梯度离心法成功分离出大鼠HSC,且活力、纯度均达到研究要求,HSC得率约为2×107/肝,细胞存活率为95%以上,纯度为90%以上。结论:本方法分离大鼠HSC合理、经济、便捷。

Objective： To established a reasonable,ecnomic and convenient method of isolating hepatic stellate cells（HSC） in rats.Methods： The method for isolation of rat hepatic stellate cells was established with D-Hanks in situ liver recirculating perfusion and then isolated perfusion with Type IV collagenase and DNaseⅠand continuous density gradient centrifugation by 10%,16% Optiprep Solution.The viability of the isolated cells was deter-mined with trypan blue staining assay.The HSC were identified by immunocytochemical staining of α-SMA.Results： the rat HSC were successfully isolated and the viability and purity were reached the requirement.The average number of HSC from a single rat liver was 2×107,with viability over 95%,purity over 90%.Conclusion： This method for isolating HSC is reasonable,ecnomic and convenient.