改良胶原酶经腹主动脉灌注消化法分离大鼠肝细胞

Separation of rat hepatocytes using modified collagenase perfusion via the abdominal aorta

背景:良好的分离技术是获取高活性肝细胞的前提。目前,国内外普遍采用的是经门静脉的两步胶原酶灌注法。但该方法仍存在胶原酶用量大、操作繁琐、流程长、对设备要求较高等问题。目的:寻找一种简单有效的大鼠肝细胞分离培养方法。方法:取SD大鼠10只,按改良经腹主动脉灌注法分离培养大鼠肝细胞,重复10次分离肝细胞实验,观察各项指标结果并与已发表文献进行对比分析。以SD大鼠作肝细胞供体,采用Ⅳ型胶原酶经腹主动脉灌注,供体肝脏肝门部结构﹑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200目和300目筛滤过,滤过后的悬液转移至离心管中分别以1000,500,300r/min离心各3min以纯化肝细胞,以锥虫蓝染色法测细胞活性,在倒置显微镜下观察肝细胞纯度及形态变化。结果与结论:胶原酶消化法所获取的肝细胞纯度高、形态完整、活性高。提示改良胶原酶经腹主动脉灌注消化法是一种较好的肝细胞分离方法。

BACKGROUND：Good separation technique is the basis of harvesting hepatocytes with high viability.At present, two-step collagenase perfusion method via portal vein is extensively used in the world.However, this method has some problems, such as high dose of collagenase, complicated operation, long process and high requirement for the instrument.OBJECTIVE：To investigate a simple effective method of separation and primary culture of rat hepatocytes.METHODS：A total of 10 Sprague Dawley rats were obtained to harvest hepatocyte by modified perfusion method through the abdominal aorta.Hepatocyte tests were repeated ten times to observe results of each index and to compare with published literatures.SD rats served as hepatocyte donors using Ⅳ type collagenase perfusion via the abdominal aorta.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 200-mesh and 300-mesh sieves, the suspension was transferred to a centrifuge tube, and then purified hepatocytes at 1 000, 500, 300 r/min centrifugation about respectively （each 3 minutes）.Trypan blue staining was utilized to measure cell viability.The purity and morphology of hepatocytes were observed under the inverted microscope.RESULTS AND CONCLUSION：Hepatocytes obtained by collagenase digestion were pure, intact and have a very good viability.The modified collagenase perfusion through the abdominal aorta was a better method for isolation of hepatocytes.