论文部分内容阅读
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 techniques are a prerequisite for obtaining highly active hepatocytes. Currently, commonly used at home and abroad through the portal vein collagenase perfusion method. However, this method still exists a large amount of collagenase, tedious operation, long process, higher equipment requirements and other issues. Objective: To find a simple and effective method of rat liver cell isolation and culture. Methods: Ten Sprague-Dawley rats were randomly divided into three groups: experimental group (n = 10), experimental group (n = 10) and control group (n = 10). SD rats were used as donors of hepatocytes, and hepatic cells were obtained by percutaneous aorta perfusion with type IV collagenase, hepatic portal structure of donor liver, hepatic and hepatic inferior vena cava were isolated by collagenase digestion, 300 mesh sieve. After filtration, the suspension was transferred to a centrifuge tube and centrifuged at 1000, 500 and 300 r / min for 3 min respectively to purify the hepatocytes. The cell viability was measured by trypan blue staining. The purity of the hepatocytes was observed under an inverted microscope And morphological changes. RESULTS AND CONCLUSION: The hepatocytes obtained by collagenase digestion had high purity, integrity and activity. Prompted modified collagenase by abdominal aorta perfusion digestion is a better method of liver cell isolation.