人脐带血间充质干细胞的体外培养及其影响因素(英文)  

In vitro culture and influencing factors of mesenchymal stem cells derived from human umbilical blood

作　　者：黄伟[1] 祝建中[1] 苗宗宁[1] 王玲[1] 王新[1] 

机构地区：[1]无锡市第三人民医院组织工程和细胞生物学研究室,江苏省无锡市214041

出　　处：《中国组织工程研究与临床康复》2007年第3期572-575,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:多潜能间充质干细胞在特定的培养条件下可以分化为骨、脂肪、软骨、肌肉以及内皮细胞,是目前倍受关注的一类具有多向分化潜能的组织干细胞。目的:建立人脐带血来源的间充质干细胞体外培养、扩增的方法,并分析其影响因素。设计:随机对照实验。单位:无锡第三人民医院组织工程和细胞生物学研究室。材料:健康新生儿脐带血(来源于无锡第三人民医院妇产科临产室,均征得产妇及其家属同意)70例,每例40mL;低糖基本必需培养基(Gibco),胎牛血清(Gibco),青链霉素(Gibco),胰蛋白酶(Gibco),FITC标记的小鼠抗人CD29、CD105、CD106(Ancell)单克隆荧光抗体和PE标记的小鼠抗人CD34、CD44、CD45、CD19、HLA-DR(Immunotech)单克隆荧光抗体,Ficoll分离液(Pharmacia)。方法:实验于2005-02/2005-12在无锡第三人民医院组织工程和细胞生物学研究室完成。①脐血间充质细胞的分离培养:健康新生儿脐带血共70例肝素(25u/mL)抗凝,分离单个核细胞,其中60例沉淀细胞用细胞培养液(低糖基本必需培养基+50g/L胎牛血清+10g/L青链霉素)重悬,另10例用高糖基本必需培养基重悬(其余培养条件相同)。细胞长到80%融合时,以1.0×107个/L的密度接种于培养瓶中进行扩增培养。②胎牛血清包被对人脐血间充质干细胞贴壁率的影响:取生长状态良好、纯化后对数生长期的人脐血间充质干细胞,分为血清包被组和无血清包被组,分别观察其贴壁率。③间充质干细胞的形态学和生长曲线:在相差显微镜(OLYMPUS CK40)下观察细胞生长状况,数码成像系统(OLYMPUS DP50)摄像记录。④免疫表型:取扩增第5代的脐血间充质干细胞,用EPICS-ALTRA流式细胞仪进行检测。主要观察指标:①观察高糖组和低糖组细胞的生长状态。②观察不同时间点胎牛血清包被组与未包被组细胞的贴壁情况并分别计算贴壁率。结果:本实验总共培养和分析了70例脐血BACKGROUND： Multipotantial mesenchymal stem cells （MSCs） can be differentiated into bone, fat, cartilage, muscle and endothelial cell at the specific conditions, so it draws great attentions of the related study. OBJECTIVE： To establish the method of in vitro culture and expansion of human umbilical blood-derived MSCs and in- vestigate their influencing factors. DESIGN： Randomized and controlled trials SETTING ： Department of Tissue Engineering and Cell Biology in Wuxi Third People＇s Hospita MATERIALS： Seventy cases of human umbilical cord blood （HUCB） of healthy neonates （selected from Parturi- ent Room, Department of Gynecology and Obstetrics, Wuxi Third People＇s Hospital, with the informed consents of the puerpera and their relatives）, 40 mL each; dulbecco＇s minimal essential medium （DMEM） of low glucose （LG） at the dose of 50 g/L, fetal bovine serum （FBS）, mycillin and trypsin （all were from Gibco）, FITC-la- beled mice anti-human CD29, CD105 and CD106 （Ancell） monocIonal fluorescent antibody, PE-labeled mice anti-human CD34, CD44, CD45, CD19, HLA-DR （Immunotech） monoclonal fluorescent antibody, Ficoll separation medium （Phatmacia）. METHODS： The experiment was carried out in Department of Tissue Engineering and Cell Biology in Wuxi Third People＇s Hospital from February to December in 2005. （1）lsolated culture of MSCs： Totally 70 cases of the healthy neonatal cord blood were anticoagulated by 25 u/mL heparin, and 60 cases of isolated HUCB mononuclear cell were precipitated and suspended by cell culture fluid （50 g/L DMEM-LG ＋ 50 g/L FBS ＋ 10 g/L mycillin）, while oth- er 10 cases were purified with DMEM of high glucose （100 g/L） to produce adherent layer. Once the cells reached 80% confluency, they were inoculated in culture flask at the density of 1.0×10^7 cells/L for proliferative culture.（1）Influences of FBS coating on adhesive rats of MSCs： Some of the purified HUCB MSCs at logarithmic phase were coated with FBS, whereas oth

关 键 词：胎血 间质干细胞 细胞学 细胞培养技术 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]