【摘要】 目的:研究人骨髓和脐血间充质干细胞(MSC)的体外培养特性及建立细胞库的可行性。方法:Ficoll离心剂法分离人骨髓及脐血来源单个核细胞,进行体外培养并大量扩增,观察细胞生长特性,流式细胞术检测MSC表面分子标志物表达情况。结果:骨髓样本比脐血标本的MSC更易于体外短期培养扩增,骨髓样本(5×106)体外培养2周约获得MSC3×107～1.2×108,成功率达100%,而脐血样本的MSC较难培养,成功率低(12.9%),细胞数扩增有限。对冻存复苏前后的细胞进行流式细胞术检测提示MSC表面标志物CD29、CD44、CD59、CD90、CD105、CD166等均阳性表达,造血细胞标志物CD34、CD14和CD45为阴性表达,提示MSC对本室的冻存复苏程序耐受能力较好,可获得复苏后扩增培养。结论:本研究表明对成人骨髓MSC建库的条件较成熟,获得成功培养的MSC分子表型在冻存复苏前后无明显改变。而脐血来源的MSC培养条件有待改善方能提高MSC培养成功率。更多还原

【Abstract】 Objective To study the characteristics of in vitro expansion of mesenchymal stem cells (MSC) from the bone marrow and umbilical cord blood and feasibility of establishment of MSC bank. Methods Mononuclear cells (MNCs) from human bone marrow and umbilical blood were separated by Ficoll and cultured in vitro, achieving significant expansion. Cell growth characteristics were observed and surface markers were detected by flow cytometry. Results MSC from human bone marrow samples were more easily expanded in vitro than those from umbilical cord blood. For bone marrow samples, in vitro two weeks 5× 106 MSC could be propagated to the amount of 3× 107～ 1.2× 108. And cultures of all samples could be 100% obtained. While MSC from umbilical cord blood were successfully cultured at low rate of 12.9% . Surface markers assay showed that, whether before or after cryopreservation, CD29, CD44, CD59, CD90, CD105 and CD166 were all positively expressed and hematopoietic markers CD34, CD14 and CD45 were all negatively expressed. All cell samples after cryopreservation gained further expansion in vitro, which indicated that MSC had good tolerance to the cryopreservation procedures. Conclusions These results suggest that establishment of cell bank of adult human bone marrow MSC is relatively in a mature status. Surface markers expression were not affected by the standard cryopreservation and thawing procedures. Culture and expansion conditions of MSC from umbilical cord blood samples should be further studied and improved.更多还原