人骨髓间充质干细胞玻璃化冷冻复苏后的生物学活性  

BIOLOGICAL ACTIVITY OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS AFTER VITRIFICATION FROZEN AND THAW

作　　者：王延伟[1] 沈肖方[1] 王跃嗣[2] 张玉花[1] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院生殖中心实验室,山东青岛264000 [2]滨州医学院生物技术教研室

出　　处：《青岛大学医学院学报》2013年第1期33-34,37,共3页Acta Academiae Medicinae Qingdao Universitatis

基　　金：烟台市科技计划资助项目(2008142-11)

摘　　要：目的探讨人骨髓间充质干细胞(BMSCs)玻璃化冷冻复苏后细胞的存活率和诱导分化能力。方法体外分离纯化人BMSCs,流式细胞仪检测其纯度。应用玻璃化冷冻方法进行冷冻保存,复苏后台盼蓝染色鉴定存活率。在DMEM/F12培养的BMSCs中加体积分数0.10的胎牛血清和10μg/L重组人碱性成纤维细胞生长因子预诱导2d,再加0.1μmol/L全反式维甲酸和10μg/L胶质细胞系源性神经营养因子进行正式诱导3d,免疫荧光染色检测Nestin、微管相关蛋白(MAP2)、髓鞘碱性蛋白(MBP)、胶质纤维蛋白(GFAP)的表达。结果 BMSCs强表达CD44和CD90,不表达CD34和CD45。玻璃化冷冻解冻后细胞存活率>95%。经诱导后,BMSCs强阳性表达MBP和GFAP,弱阳性表达Nestin和MAP2。结论玻璃化冷冻复苏后的人BMSCs存活率高并能成功诱导为神经胶质细胞。Objective To study the post-thaw survival rate and differentiation ability of human bone marrow mesenchy- real stem cells （BMSCs）. Methods Human BMSCs were isolated and purified in vitro, its purity was detected by flow cytome- ter. The cells were offered cryopreservation by using vitrification frozen. The survival rate was measured after thawing. BMSCs were then cultured in DMEM/F12 supplemented with 10 μg/L bFGF and 10% fetal bovine serum pre-induce for two days, and then induced by RA （0.1 μmol/L） and 10 μg/L Glial cell line derived neurotrophie factor （GDNF） for formal induction for three days. the expressions of Nestin, MAP2, MBP, GFAP were detected by immunofluorescence staining. Results Strong expres- sions of CD44 and CD90 were noted in BMSCs, but no CD34 and CD45; post-thaw cell survival was 〉95 %. After induction, the expressions of BMSCSMBP and GFAP were strongly positive, and that of Nestin and MAP2 weakly positive. Conclusion The survival rate of post-thaw BMSCs is high and can be successfully induced to become neuroglial ceils.

关 键 词：间质干细胞 骨髓 玻璃化冷冻 细胞分化 神经胶质 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]