胰酶消化时长对GFP转基因小鼠BMSC传代生长特性的影响  

Effect of trypsinization duration on passaging and growth characteristics of BMSCs from GFP transgenic mice

作　　者：和法莲 白盈盈[1] 陈慧芬[1] 李自安 杨建勇 潘兴华[1] 刘高米洋 

机构地区：[1]干细胞与免疫细胞生物医药技术国家地方联合实验室,云南省细胞治疗技术转化医学重点实验室,昆明医科大学昆明总医院临床学院,昆明650032

出　　处：《西南国防医药》2018年第1期25-28,共4页Medical Journal of National Defending Forces in Southwest China

基　　金：全军实验动物专项面上项目(SYDW2016004);云南省科技创新团队建设项目(20140810)

摘　　要：目的比较胰酶消化时间对绿色荧光蛋白转基因小鼠骨髓间充质干细胞(BMSC)传代生长、分化能力的影响。方法取含相同细胞量的P2代绿色荧光蛋白转基因小鼠BMSC悬液接种于6孔细胞培养板中,待细胞生长融合度达到80%~90%时,用0.25%的胰酶消化5、10、15和20 min,比较不同时间点胰酶的消化能力、再次贴壁生长状态、培养上清中BMSC死亡碎片的数量以及诱导成脂、成骨、成软骨分化能力。结果消化5 mins后,大量BMSC处于贴壁状态,无法传代;消化10、15 mins后,80%以上的BMSC缩成圆形并脱落,二次贴壁后呈长梭形,表面光滑,具有多向分化潜能;消化20 min后,BMSC全部悬浮,再贴壁后呈宽大扁平状,表面粗糙,培养上清中悬浮大量细胞死亡碎片。结论绿色荧光蛋白转基因小鼠BMSC传代培养中,0.25%胰酶的最佳消化时间为10~15 mins。Objective To compare the effects of the trypsinization duration on the passaging, growth and differentiation capabilities of bone marrow-derived mesenchymal stem cells （BMSCs） from green fluorescent protein （GFP） transgenic mice. Melhods The solution of Passage 2 （P2） mouse BMSCs from GFP-transgenic mice was inoculated on a 6-well cell culture plate, with equal number of cells in each well. When these cells reached to 80%-90% confluency, 0.25% trypsin was used to digest cells for five mins, 10 mins, 15 mins and 20 mins; and then, the digestive capability of trypsin, the state of adherent cell growth again, the number of fragments of dead BMSCs in the supernatant, and the capability of induced adipogenic, osteogenic and chondrogenic differentiations at each of these points of time were compared, Results Five rains after digestion started, a large number of BMSCs were adherent and could not be p assaged; 10 and 15 mins after digestion started, above 80% BMSCs were rounded and detached, and cellsbecoming adherent again grew into spindle shape, with smooth surface and with multi-potential differentiation capability; 20 rains after digestion started, all BMSCs were floating, ceils becoming adherent once again grew into flat shape with rough surface, and large number of fragments of dead cells floated in the supernatant. Conclusion During the passaging of BMSCs from green fluorescent protein （GFP） transgenic mice, the optimal digestion duration of 0.25% trypsin is 10-15 mins.

关 键 词：绿色荧光蛋白 小鼠 骨髓间充质干细胞 胰酶消化 时间 

分 类 号：R318[医药卫生—生物医学工程]