胶原酶及灌流时间对大鼠肝细胞分离效果的影响  

The effect of collagenase and duration of liver perfusion on isolation of rat hepatocytes

作　　者：于聪慧[1] 冷希圣[2] 魏玉华[2] 刘继超[2] 杜如昱[2] 

机构地区：[1]北京军区总医院外科,100700 [2]北京大学人民医院外科

出　　处：《中华实验外科杂志》2000年第6期541-542,共2页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金国际合作项目!(1997年特别资助项目 )

摘　　要：目的　研究肝细胞分离方法 ,提高肝细胞分离质量。方法　用两种质量分数均为0 .0 5 %胶原酶溶液 (Ⅰ和Ⅳ型 )经门静脉灌注肝脏 ,分离大鼠肝细胞。分别于 5、10、15min灌流肝脏 ,观察对肝细胞活率及产量的影响。将肝细胞溶液用质量分数为 0 .0 2 %胶原酶溶液孵育 10min ,观察孵育对肝细胞活率的影响。结果　Ⅳ型胶原酶消化效果明显优于Ⅰ、Ⅳ型和Ⅰ型 ,灌流 10min细胞存活率分别为 (89.5± 3 .5 ) %和 (5 8.0± 14.0 ) % ,而产量分别为 (2 .5± 0 .2 )× 10 11个 /L和 (0 .9± 0 .5 )× 10 11个 /L ,差异有非常显著性 (P <0 .0 0 1) ,细胞培养及细胞组织化学证实分离细胞的结构和功能正常。低浓度的胶原酶孵育能明显提高肝细胞的活率。结论　Ⅳ型胶原酶可获得良好的肝细胞分离效果。胶原酶孵育能提高细胞活率。Objective To study the method of hepatocyte isolation.Methods Using two defferent collagenases (0.05%,type Ⅰ and Ⅳ) the rat hepatocytes were isolated. The effects of defferent types of collagenases and different duration of liver perfusion(5,10 and 15 min) on viability and outputs of hepatocytes were investigated and the effect of culture on viability in 0.02% collagenase solution studied. Results The best results of isolation were accessed by collagenase typeⅣ.Cell viability of two groups(Ⅳ and Ⅰ) was (89.5±3.5)% and (58.0±14.0)%,output (2.5±0.2)×10 11 cell/L and (0.9±0.5)×10 11 cell/L following 10 min perfusion (P<0.01) respectively. The structure and functions were normal by culture and biochemical assay.The study also showed that cell viability was increased by the method of cultue in low concentration of collagenase solution.Conclusion Collagenase type Ⅳ and 10 min liver perfusion were the best conditions for rat hepatcyte isolation.

关 键 词：肝细胞分离 胶原酶 灌流时间 大鼠 

分 类 号：R363[医药卫生—病理学]