脐血间充质干细胞的体外培养及其表面标志变化

In vitro culture and surface marker variations of umbilical cord blood mesenchymal stem cells

背景:目前有关脐血间充质干细胞的体外培养和大规模扩增方法不一,存在一定困难。目的:观察脐血间充质样干细胞体外分离和培养的方法,并检测其表面分子的变化。方法:取新鲜采集脐带血,用1.077g/cm3的淋巴细胞分层液,密度梯度离心法分离脐血单个核细胞。将脐血单个核细胞接种于37℃、含体积分数为5%CO2培养箱内培养。于不同时间观察细胞形态的变化并通过流式细胞仪检测细胞表面分子的表达情况。结果与结论:从脐血中分离出的单个核细胞,培养中先出现大量的造血细胞集落,CFU-GM与BFU-E集落形成最多,集落分别增加了(37.1±2.3)和(10.4±1.7)倍,瑞氏染色显示这些细胞大多数为粒系的集落(80.1±85.2)%,其次为红系的细胞集落(14.2±1.8)%,7d后出现贴壁的扁平状上皮样细胞和长梭形成纤维样细胞,同时有大量的破骨样细胞混杂。扩增后第14天经流式细胞仪分析CD38+细胞为1.64%,CD34+/CD38+细胞为1.71%,CD34+/CD38-细胞为0.55%,PI+细胞为0.05%,Annexin-V+细胞为0.18%。随着培养时间的延长,细胞数目不断增加,培养21d时,单个核细胞扩增了近7.8倍.,第28天增加了1.71倍。经流式细胞仪分析CD38+细胞为74.32%,CD34+/CD38+细胞为1.61%,CD34+/CD38-细胞为0.24%。提示脐血间充质干细胞可以体外培养。

BACKGROUND：Currently,there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells（UCB-MSCs）.OBJECTIVE：To investigate the isolation,purification and culture of UCB-MSCs in vitro,and to detect its surface marker variation.METHODS：The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation,followed by incubation in an incubator containing 5%CO2 at 37 ℃.The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry.RESULTS AND CONCLUSION：The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones,most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid,increasing by（37.1±2.3） and（10.4±1.7）,respectively.Switzerland staining showed most of them were granulocyte clones（80.1±85.2）%,next was erythroid clones（14.2±1.8）%.At 7 days after culture,some shuttle fibroblast-like cells and flat osteogenic-like cell spread the whole plastic well.At 14 days after culture,flow cytometry showed CD38＋ cells accounted for 1.64%,and CD34＋/CD38＋ cells accounted for 1.71%,and CD34＋/CD38-were 0.55%.PI＋ and Annexin-V＋ cells accounted for 0.05% and 0.18% respectively.At 21 days after culture,CD38＋,CD34＋/CD38＋ and CD34＋/CD38-cells were 74.32%,1.61%,and 0.24%.The results reveled that UCB-MSCs can be isolated and cultured in vitro.