一种同时分离肝细胞和储脂细胞的简易方法  

An efficient method for hepatocytes and fat-storing cells isolation at one time

作　　者：于聪慧[1] 林梅[1] 杨荣华[1] 姚军波[1] 吕民生[1] 

机构地区：[1]北京军区总医院肝胆外科,100700

出　　处：《中华实验外科杂志》2004年第10期1179-1180,共2页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目 (30 1 70 4 2 1 )

摘　　要：目的　探讨同时分离肝细胞和储脂细胞的有效方法。方法　采用门静脉胶原酶灌注消化法同时分离大鼠肝细胞和储脂细胞 ,先用 3 0 0r/min(2 0℃ )低速离心 5min ,将细胞悬液分成上清和沉淀两部分并分别放入两个离心管中。A管为沉淀部分 (肝细胞悬液 )经 0 .0 2 %胶原酶孵育液孵育 10min(3 7℃ 5 %CO2 ) ,10 0目网过滤 ,离心后即为肝细胞。B管为上清部分主要是储脂细胞 ,经胶原酶恒温震荡消化 (2 0 0r/min ,3 7℃ ) ,2 0 0目网过滤 ,Nycodenz液进行梯度离心 (3 0 0 0r/min)后即得储脂细胞。结果　肝细胞存活率和细胞产率分别为 (93 .5± 3 .5 ) %和 (2 .4± 0 .2 )×10 8/大鼠 ,储脂细胞分别为 (94.5± 3 .5 ) %和 (2 .3± 0 .4)× 10 7/大鼠。肝细胞组织学及组织化学检测肝细胞形态正常 ,细胞培养功能表达正常。储脂细胞在激发波长为 3 2 8nm的荧光显微镜下发出蓝绿色荧光 ,细胞培养贴壁良好。结论　一步法同时分离肝细胞和储脂细胞是一种简单有效的方法。Objective To study the efficient method to isolate the hepatocytes and fat-storing cells.Methods The hepatocytes and fat-storing cells were digested in one step through the portal vein collagenase perfusion;the cell solution was isolated into two parts by low speed centrifugation (300 r/min,5 mins).The suppurate,which was rich of the fat-storing cells,was re-digested by collagenase and vibration,the cells were obtained by ladder centrifugation by Nycodenz solution (3?000 r/min).The deposition,which was rich of hepatocytes,was cultured by the collagenase and obtained by low speed centrifugation.Results The survival rate and output of the hepatocytes were (93.5± 3.5)% and (2.4± 0.2)× 10 8/rat;fat-storing cells were (94.5± 3.5)% and (2.3± 0.4)× 10 7/rat,respectively.Hepatocytes and fat-storing cells had normal structure and function.Conclusion The one step isolation method was an efficient method in hepatocytes and fat-storing cells isolation.

关 键 词：分离方法 肝细胞 储脂细胞 门静脉胶原酶灌注消化法 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]