儿童骨髓间充质干细胞体外培养与富集效率的研究

Study on Cultivation in vitro and Enriching Efficiency of Bone Marrow Mesenchymal Stem Cells in Children

目的比较密度梯度离心法和全骨髓培养法分离培养儿童骨髓间充质干细胞(BMSCs)获得的细胞纯度与克隆形成效率。方法取等量抗凝的儿童骨髓分别以密度梯度离心法和全骨髓培养法分离培养BMSCs,传代至第3代分别计数2种方法所得BMSCs的细胞总数,流式细胞仪检测表面标记物及其纯度,对第5代BMSCs分别绘制生长曲线,全骨髓培养法培养至第6代流式细胞仪再检测表面标记物及其纯度。结果2种方法均能有效培养出BMSCs,BMSCs强表达CD105及CD13,低表达CD34;3mL骨髓样本用全骨髓培养法扩增至第3代平均可获得3.15×107个细胞,而密度梯度离心法3mL骨髓只获得1.08×107个细胞,二者有显著性差异(P<0.05);密度梯度离心法第3代BMSCs纯度达95%以上,全骨髓培养法第3代BMSCs纯度约85%,多次换液传至第6代纯度可达到95%以上;2种方法第5代BMSCs生长曲线近似,接种后0～3d为生长潜伏期,3～6d为对数生长期,之后进入平台期,无显著性差异(P>0.05)。结论密度梯度离心法分离培养BMSCs,可在短时间内获取纯度较高的BMSCs;全骨髓培养法分离培养BMSCs,可从少量标本获得最大数量的BMSCs,多次换液传代也可获得纯度较高的BMSCs。

Objective To determine the difference between density- gradient centrifugation and full- bone -marrow culture method for the isolation and cultivation of bone marrow mesenchymal stem cells（BMSCs） ,and provide basis for selecting the best method for different experimental purpose. Methods Bone marrow aspirate were harvested and cultured with density - gradient centrifugation and full - bone - marrow culture method respectively, the third passage cells from 2 methods were used to detect the specific surface markers with Flow cytometry and the total number of all cells were calculated ;the fifth passage cells were cultured for another days to draw the growth curve. The sixth passage ceils from full - bone - marrow culture method were detected the surface marker as before. Results The 2 methods were all effective to harvest BMSCs from bone marrow, which expressing CD105 and CD13 strongly, but no CD34 ;3 mL bone marrow aspirate can generate 3.15 × 10^7 BMSCs with full - bone - marrow culture, but just 1.08 × 10^7 BMSCs with density - gradient centrifugation. There was much difference in 2 methods （ P 〈 0.05 ）. And there was no significant difference in growth curve of the Fifth passage cells from 2 culture methods. The purification of the third passage ceils with density - gradient centrifugation was 95% ,and that with full - bone - marrow culture was about 85% ,but that was more than 95% when the ceils cultured to the sixth passage. The growth curves of the fifth generation BMSCs based on the 2 different methods appear to be similar. 0 - 3 d after vaccination was the latent growth period and 3 -6 d was the logarithmic growth period. Then it was the period for growth into platform. It showed no significant statistical differences （ P 〉 0.05 ）. Conclusions The method of density - gradient centrifugation is applicable to get more purified BMSCs within short time, and the full - bone - marrow culture method was suited to get much more BMSCs with little bone marrow aspirate. It can also get more purified BMSCs by changing culture fluid and going down to posterity for many times.