CD34＋CD38-Lin-细胞群单个细胞培养的初步研究

A Preliminary Study on the Culture of Single Hematopoietic Stem Cell

目的探讨单个CD34+CD38-Lin-细胞在体外培养体系中的生存和增殖能力。方法用免疫磁珠从脐带血单个核细胞中分选出Lin-细胞,然后用流式细胞仪筛选出CD34+CD38-Lin-细胞,并由流式细胞仪将单个CD34+CD38-Lin-细胞直接放入培养孔中进行培养。培养基中分别加细胞因子Shh、BMP-4或Jagged-1作为实验组,未加细胞因子的以等量PBS作为对照。应用细胞计数法、集落培养计数法检测不同培养条件下各组细胞数量的变化及集落形成能力的差异。结果培养第3天,各组均能见到单个细胞的分裂倍增现象。培养1周后,各实验组的细胞存活率均高于对照组,顺序为Jagged-1组>BMP-4组>Shh组>对照组,而每孔细胞数的顺序为Jagged-1组>BMP-4组>对照组;实验组中每孔细胞数为0的孔数明显低于对照组,尤其是Jagged-1组,而实验组每孔细胞数多于17的孔数高于对照组,其中BMP-4组最高。实验组的每孔集落数明显高于对照组,实验组之间的差异并不明显。另外,单个细胞集落培养后能形成红系爆式集落及粒细胞、单核细胞、粒-单核细胞集落形成单位等各式集落。结论单个CD34+CD38-Lin-细胞能够单独完成生存、自我更新及增殖等过程,而添加细胞因子Shh、BMP-4、Jagged-1能够增强其上述能力,显示微环境的重要性。单个CD34+CD38-Lin-细胞只能形成一种细胞集落,说明微环境对于维持CD34+CD38-Lin-细胞群的全能性必不可少。

To investigate the biological behavior including survival and proliferation of CD34 ＋ CD38 Lin cells when they are cultured at single cell level. Methods Purified umbilical cord blood CD34 ＋ CD38 Lin cells were separated at single cell level in 96well plates using flow cytometry for four goups ： control group （ CD34 ＋ CD38 Lin cell plus stem cell medium）, Shh group （ CD34 ＋ CD38 Lin cell plus stem cell medium and Shh） , BMP-4 group （ CD34 ＋ CD38 Lin cell plus stem cell medium and BMP-4） , Jagged-1 group （ CD34 ＋ CD38 Lin cell plus stem cell medium and Jagged-1 ）. Methylcel lulose medium was used in the colonyforming experiment which was also in four groups as previously. The number of cells and colonyforming units in each well for the four groups was evaluated at different time points（ day 1, 3,7 ） with fluorescence microscopy counting method. Results Division of single cell was observed to be amplified in all of these groups from day 3. And meanwhile, after 1-week culture, the survival rates for the treated groups were all higher than the control group （JaggedI group 〉 BMP-4 group 〉 Shh group 〉 control） , while the cell number in each well was also highest in the Jagged-1 group （ Jagged-1 group 〉 BMP-4 group 〉 control）. The number of wells with a cell number of zero was significantly fewer in all treated groups （ especially the Jagged-1 group） than in the control group; meanwhile, the number of wells with a cell number higher than 17 was evidently higher in all the treated groups （especially the BMP-4 group） more than controls. Colonyforming units for erythroid （BFUE）, granulocyte （CFUG）, macrophage （CFU-M）, and granulocyte macrophage （CFUGM） were observed for all of these experimental groups, and there was no significant difference between the four experimental groups. Conclusions CD34 ＋ CD38 Lin cell can achieve the survival, selfrenewal and proliferation when cultured at single cell level, and the adding of Shh, BMP4, and Jagged-1 can enhance such capabilities. However, CD34 ＋ CD38 Lin cell can only maintain cell totipotency in its niche.