小鼠肝脏原位持续灌流方法的建立及其在肝脏非实质细胞分离中的应用

目的建立一种小鼠肝脏原位持续灌流的方法,并将该法应用于肝脏非实质细胞的分离。方法利用三通管和静脉留置针将门静脉、蠕动泵软管以及下腔静脉连接形成封闭回路,自门静脉端注入37℃的胶原酶消化液约60 ml,调节蠕动泵转速为20 ml.min-1行循环灌注约30 min,摘除肝脏剪切成小块组织,加入胶原酶孵育液消化30 min后碾碎制备成细胞悬液,离心后取沉淀先后以50%和35%的Per-coll密度离心即可获得小鼠肝非实质细胞。结果肝脏非实质细胞的得率为0.5×107～1×107个/鼠肝,流式检测CD45+细胞占(27.21±1.47)%。结论胶原酶肝脏原位持续灌流+体外孵育消化结合Percoll密度离心是分离小鼠肝脏非实质细胞的可靠方法。

Isolation and purification of murine hepatic non-parenchymal cells by liver circulatory perfusion in situ

Aim To establish a new method of liver perfusion in mice for isolation and purification of hepatic non-parenchymal cells.Methods The entire mouse abdominal cavity was exposed,portal vein and inferior vena cava were cannulated with intravenous catheters,then they were connected with three-way pipe and flexible tube to form a closed loop.Immediately after flushing liver,the digestion began with 60 ml collagenase Ⅳ in the circulatory loop at a flow rate of 20 ml.min-1 for up to 30 min.The liver fragments were pressed and cells were collected after incubation in collagenase solution for 30 min.Cell pellets were resuspended in 50% /35% isotonic Percoll solution to purify nonparenchymal cells.Results The yield of hepatic non-parenchymal cells per mouse was 0.5 × 107 ～ 1 × 107 and the percentage of CD45+ cells was(27.21 ± 1.47) %.Conclusion Liver collagenase perfusion in situ and Percoll density gradient centrifugation is an effective method in isolation and purification of hepatic nonparenchymal cells in mice.