一种简易高效的大鼠原代肝细胞分离方法

目的:寻找一种简易高效的大鼠原代肝细胞分离方法。方法:在Seglen两步胶原酶灌注法的基础上加以改进,按门静脉灌注法分离培养大鼠肝细胞,重复10次分离肝细胞实验,观察各项指标结果。采用胰酶、Ⅳ型胶原酶经门静脉灌注,大鼠肝脏肝门部结构、肝上及肝下腔静脉封闭保留胶原酶消化分离获取肝细胞,经80目和200目的筛网依次过滤细胞后,细胞悬液分别以1 000、500、300 r/min离心各5 min以纯化肝细胞,以台盼蓝排染法测定细胞活性,HE染色鉴定肝细胞纯度。结果:平均每只成年Wistar雄性大鼠可以获得(1.53±0.31)×10~8个肝细胞,平均获得肝细胞活率可达到94.63%,平均获得肝细胞纯度为96.7%。结论:本实验介绍的方法简便易行,且肝细胞产量较高,活力好,纯度高,适宜在一般条件的实验室推广和应用。

A facility and high efficiency separation method of rat primary hepatocytes

Objective:To search a facility and high efficicenty separation method of rat primary hepatocytes.Methods:Rat hepatocytes were isolated and purified by using the modified Seglen' s two-step method，portal vein perfusion.Hepatocyte tests were repeated about ten times to observe.Rats served as hepatocyte donors using Ⅳ type collagenase and pancreatin via the portal vein.Liver hepatic portal structures，the upper and inferior vena cava of the donors were closed to retain collagenase digestion to obtain hepatocytes.Following filtration through 80-mesh and 200-mesh sieves，the suspension was transferred to a centrifuge tube，and then hepatocytes were purified at 1 000，500，300 r/min centrifugation about respectively (each 5 minutes).The cell viability and purity were identified by trypan blue and HE stain.Results:The average yield of hepatocytes were （1.53±0.31）×108 cells per rat with an average viability of 94.63%，and the purity was 96.7%.The hepatocytes showed as oval，roundness and polygon with HE stain，and there was one or two circular nuclei in each cell.Conclusion:The separation method of hepatocytes was simple and easy，with advantages of high yield good viability and high purity，so it would be applied widely in ordinary laboratory.