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

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例脐血间充质干细胞样本。由于10例予含高糖的培养基培养的标本均未获得理想的间充质干细胞,故未进行统计学分析。①按照本实验所建立的培养体系,约20%的样本成功培养出脐血间充质干细胞。原代细胞在培养2周后可达到融合,一般其倍增时间为3～4d,传代后7～8d即可达到融合。②扩增至第5代的间充质干细胞结果显示,脐血间充质干细胞均一稳定地表达间充质干细胞相关的抗原标记:CD29,CD44,CD105,但不表达CD34,CD45,CD106,HLA-DR,这与源于骨髓的间充质干细胞的表面抗原标记相一致。③血清包被组间充质干细胞贴壁率显著高于无血清包被组(P<0.01)。结论:脐带血中的间充质干细胞可在体外培养、扩增,能够作为间充质干细胞的一种有效来源。高糖可能抑制人脐血间充质干细胞的生长和扩增。

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 others were not, and the adhesive rate was observed in two groups.（3）Morphology and growth curve of MSCs： The growth of cells were observed by phase contrast microscope （OLYMPUS CK40）, and digital imaging system （OLYMPUS DP50） was applied to record.（4）Immunophenotype： HUCB MSCs at passage 5 were detected by EPICS-ALTRA flow cytometry. MAIN OUTCOME MEASURES ： （1）The growth of MSCs in DMEM-LG or DMEM-high glucose.（2）The adherence of MSCs with FBS coating and without FBS coating at the different time points, and their adhesive rates. RESULTS： All 70 cases of HUCB MSCs were adopted in this study, however, 10 samples failed to obtain the ideal MSCs in DMEM of high glucose and not involved in the final statistical analysis. （1）About, 20% samples succeeded to obtain the cord blood MSCs after culture. During 2 weeks of culture, primary cells began to double for 3-4 days and reached confluency at 7-8 days after passage.（2）MSCs at passage 5 stably expressed several MSCs-related antigens： CD29, CD44 and CD105, but did not express antigens CD34, CD45, CD106 and HLA-DR, which was identical to surface labeling of human bone marrow-derived MSCs. （3）The MSCs coated with FBS displayed a significantly higher adhesive rates than those without FBS coating （P 〈 0.01）. CONCLUSION： MSCs in HUCB can be cultured and expanded in vitro, and are regarded as an alternative source of MSCs for experimental and clinical applications. Moreover, high glucose can depress the growth and proliferation of MSCs in HUCB.