大鼠原代肝星形细胞的分离与鉴定

Isolation and Identification of Primary Hepatic Stellate Cells from Rat Liver

本文通过二步原位灌注的方法分离得到大鼠肝脏,随后通过机械剪切,用0.2g/L链霉蛋白酶E,0.2g/L胶原酶Ⅳ以及0.1g/L DNaseⅠ消化和分散,筛网过滤、经130g/L Nycodenz密度梯度离心后收集肝星状细胞,经台盼蓝染色后用细胞计数仪鉴定细胞存活率,α-SMA(平滑肌动蛋白)免疫细胞化学染色法鉴定肝星状细胞纯度。结果显示,肝星状细胞的得率为每个肝脏3.5×107个细胞以上,肝星状细胞纯度为95%左右,肝星状细胞存活率为(97.0±3.2)%,满足实验需要。

Two-step in situ perfused method was adopted for isolation of hepatic stellate cells （HSCs） from rat liver. By mechanical cutting after separating in vitro,the rat liver was with 0.02% pronase E, 0.02% collagen Ⅳ and 0. 01% Dnase Ⅰ for digesting and perfusing. Cell homogenates were filtered through a cell mesh and then subjected to density gradient centrifugation in the presence of 13% Nycodenz stock solution for collecting HSCs. Cell viability was assessed by Cell counter after trypan blue exclusion staining. The purity of HSCs was identified by immunocytochemistry to detect the expression of α-SMA. The results suggested that the yield of HSCs was 3.5 × 107 per rat liver. The purity of HSCs was 95% and the survival rate of HSCs was （97.0±3.2）%. In conclusion,a stable and efficient method has been developed to isolate HSCs from rat liver for further biological studies.