摘要
目的:对传统的胶原酶灌流法加以改进,以节约分离肝细胞的实验时间和成本。方法:采用离体法,分3次从肝窦灌注含有肝素钠的D-Hanks液共150mL;用30mL终浓度为0.04%的胶原酶液(Ⅰ∶Ⅳ=1∶4)灌洗2min;去除表皮等结缔组织,过筛、离心洗涤即得肝细胞。结果:平均1g大鼠肝脏能获取7×106～8×106个肝细胞,细胞存活率为96.3%。结论:简易胶原酶灌流法的胶原酶用量是Seglen二步灌注法用量的1/16,是半原位的1/4;灌流时间约5min,是Seglen二步灌注法的1/4;半原位法也通常需要6～7min。此法具有装置简单、试剂消耗量小、操作方便快捷、实验成本低的特点,为需要周期性重复建立大鼠肝细胞模型的实验提供了一种简单快速的分离方法。
Objective： To improve the existing method of rat hepatic cell isolation by collagenase perfusion to a less expense and less time consuming operation. Methods： 50 mL of D-Hanks containing heparin sodium were perfused through porta hepatis in vitro, repeated the procedure twice, followed by perfusion with 30 mL of 0.04% collagenase （Ⅰ ：Ⅳ=1：4） for 2 min; after removal of scarfskin and other connective tissues, hepatic cells were collected via the sift-through procedure and centrifugation. Results： 7×10^6-8×10^6 hepatocytes could be harvested per gram of liver tissue; the resulted cell vitality could be reached to 96.3% in average. Conclusions： The present method cuts the collagenase use to one sixth of that by method of Seglen （a method of situ perfusion in two steps）, one fourth of that by semi-in-site perfusion techniques. It also reduce the operation time to about 5 min, compared with 20 and 6-7 min in Seglen and semi-in-site method. The present method can be applied with simple facility, lower reagent consumption, easier operation and less experimental cost. It is feasible for the labs where the repeated experiments of rat hepatic cell models are needed.
出处
《生物技术通讯》
CAS
2006年第3期392-393,共2页
Letters in Biotechnology
关键词
肝细胞
胶原酶
分离
灌流
hepatic cell
collagenase
isolate
perfusion
