一种高效、快捷、经济的小鼠胰岛细胞分离纯化方法  

An Efficient,Fast and Economic Method for Isolation and Purification of Mouse Islet Cell Masses

作　　者：徐小明[1,2] 林戈[1,2] 段馨[1] 卢光琇[1,2] 

机构地区：[1]中南大学生殖与干细胞工程研究所,中国湖南长沙410008 [2]中南大学人类干细胞国家工程研究中心,中国湖南长沙410008

出　　处：《生命科学研究》2011年第1期46-50,共5页Life Science Research

基　　金：国家重点基础研究发展计划"973"项目(2005CB522705);国家自然科学基金资助项目(30800650);湖南省自然科学基金资助项目(09JJ4010)

摘　　要：采用改良的胶原酶P胆总管内逆行灌注膨胀胰腺的分离方法,比较Ficoll-400不连续密度梯度离心法和人工挑取法两种纯化胰岛的方法,用双硫腙(DTZ)对胰岛细胞团进行特异性染色计算胰岛产量及纯度,用台盼蓝染色判定胰岛细胞活性,使用间接免疫荧光法检测体外培养7 d后胰岛的功能.采用人工挑取法平均每只小鼠可获取的胰岛细胞团(245±35.6个)明显高于密度梯度离心法(120±26.3个)(n=5,P<0.05),两种方法分离纯化的胰岛活力均大于98%;但人工挑取法获得胰岛细胞团的纯度(100%)要高于密度梯度离心法(90%);纯化胰岛所用时间,采用人工挑取法(22±4 min)也少于密度梯度离心法(31±3 min).间接免疫荧光染色检测体外培养7 d后的胰岛细胞团,两种方法分离纯化的胰岛细胞团均具有良好的功能.胶原酶P胆总管内灌注消化分离法联合人工挑取纯化胰岛细胞团的方法,是一种高效、快捷、经济的小鼠胰岛细胞团分离纯化方法.A modified method for mouse islet isolation was reported.Pancreas of mice was perfused with collagenase P via the bile duct.A comparison of two purification islets methods of density gradient centrifugation using Ficoll-400 and artifical pick,islets was then tested for their specificity,vitality and physiological function by dithiozine（DTZ）,Typan Blue and indirect immunoflurescence staining respecti-vely.The average number of islet cell masses obtained of each mouse for artificial pick method（245±35.6） was significantly higher than that of density gradient centrifugation method（120±26.3）（n=5,P0.05）.The vitality of both methods to isolate and purify islet was more than 98%.The average time of purification islet by artificial pick method（22±4 min） was less than that of density gradient centrifugation method（31±3 min）.Islets isolated by both methods have good physiological function with indirect immunofluorescence staining after cultured 7 d in vitro.The modified method which pancreas of mice was perfused with collagenase P via the bile duct and then artificial pick for islet purification is an efficient,fast and economic isolation and purification of mouse islet.

关 键 词：胰岛 分离纯化 小鼠 

分 类 号：R322[医药卫生—人体解剖和组织胚胎学]