【摘要】 目的:寻找脐带血来源的间充质干细胞体外分离、纯化及培养的条件,以建立稳定的体外培养体系,满足实验和临床的需要。方法:实验于2005-06/2006-04在青岛大学医学院附属医院儿科研究所完成。脐带血36份,50～60mL/份,取自青岛大学医学院附属医院产科健康产妇正常足月顺产或剖宫产分娩婴儿的脐带血,经产妇本人及家属同意。无菌条件下取正常足月剖宫产胎儿的脐带血,经肝素抗凝,用淋巴细胞分离液分离脐血单个核细胞。16份脐血每份分为3组,分别以间充质干细胞接种密度1×107L-1、1×109L-1、1×1011L-1接种于未包被6孔培养板内,加入5%胎牛血清的DMEM-LG培养。20份脐血每份分为3组,以1×1011L-1单个核细胞密度分别加入5%、10%和20%胎牛血清的DMEM-LG接种于胎牛血清包被的6孔培养板内培养。采用常规的间充质干细胞培养Friedenstein法进行脐血间充质干细胞的培养和扩增培养。观察不同脐血单个核细胞的接种密度、培养液胎牛血清浓度和胎牛血清包被培养皿对间充质干细胞培养的影响。结果:①胎牛血清未包被组以1×107L-1和1×109L-1接种组培养7d后贴壁数量很少,贴壁的细胞未长满瓶底即死亡,无法传代。1×1011L-1组贴壁细胞较多,间充质样细胞与破骨样细胞并存。1×1011L-1组分5%、10%、20%3种血清浓度,贴壁细胞均较多,以间充质干细胞为主,胎牛血清浓度5%组间充质干细胞的纯度较10%及20%组高,破骨样细胞相对较少;胎牛血清包被组与未包被组相比,贴壁细胞少,培养的间充质干细胞纯度较高,破骨样细胞相对较少。②流式细胞仪检测脐血间充质干细胞的免疫表型,结果显示,脐血P3代间充质干细胞不表达或极弱表达CD34、CD45、CD106等造血细胞标志,稳定地高表达CD29、CD44、CD105等间充质细胞相关的表面抗原标记物。结论:将脐血单个核细胞以1×1011L-1高密度接种在5%低胎牛血清浓度的低糖DMEM培养基中、胎牛血清包被培养瓶等条件下,可以在体外成功的培养出较纯化的脐血间充质干细胞,其培养成功率和间充质干细胞纯度较高。更多还原

【Abstract】 AIM: To explore the conditions for isolation, purification and culture of human umbilical cord blood derived mesenchymal stem cells (MSCs) in vitro, and set up a stable culture system to meet the experimental and clinical needs.METHODS: The experiment was conducted from June 2005 to April 2006 at the Research Center of Pediatrics, Medical College of Qingdao University. The umbilical cord blood (n =36, 50-60 mL per sample) was collected from full term normal deliveries or scheduled for cesarean section, which was agreed by the parturient and their relatives. The specimens were obtained sterilely with preservative-free heparin, and the cord blood mononuclear cell was isolated by lymphocyte separation medium. Each sample (n =16) was divided into 3 groups, then the MSCs with the final density of 1×107 L-1, 1×109 L-1 and 1×1011 L-1 separately were inoculated in the uncoated six-well culture plates and cultured in DMEM-LG containing 5% fetal bovine serum (FBS). In the remaining samples, each sample (n =20) was divided into 3 groups, then the mononuclear cells with the final density of 1×1011 L-1 were seeded in the six-well culture plates coated with FBS, which was supplemented with DMEM-LG containing 5%, 10% and 20% FBS, respectively. Friedenstein method was used to culture and amplify MSCs. The concentration of different cord blood mononuclear cell, concentration of FBS and effect of culture plates coated with FBS on culture of MSCs were investigated. RESULTS: ①After culture for 7 days, the MSCs in uncoated group seeded with the density of 1×107 L-1 and 1×109 L-1 had few adherent cells, which died before fully covered and could not passage. More adherent cells could be found when seeded with the density of 1×1011 L-1, and the MSCs-like cells and osteoclast existed at the same time. MSCs in coated group seeded with the serum concentrations of 5%, 10%, and 20% appeared more adherent cells, mainly MSCs. The purity of the MSCs in the 5% FBS group was higher than 10% and 20% groups, but the osteoclast were fewer; Compared with the uncoated group, the adherent cells of the coated group were fewer, but the purity was higher and the osteoclast were fewer. ②Immunophynotype of MSCs was detected with flow cytometry, which showed that P3 MSCs hardly expressed or expressed few haematopoietic cells antigens (CD34, CD45, CD106), but stably expressed MSCs-related antigens (CD29, CD44, CD105).CONCLUSION: MSCs can be cultivated successfully in vitro with high successful rate and purity when 1×1011 L-1 mononuclear cells were seeded in low-sugar DMEM culture medium containing 5% FBS and a culture flask coated with FBS.更多还原