人骨髓间充质干细胞体外分离培养方法的比较研究

目的 比较Ficoll密度梯度离心法分离人骨髓间充质干细胞(mesenchymal stem cells,MSCs)与全骨髓培养法之间的差异,以期建立一种简便、实用的基于临床移植需要的MSCs分离方法.方法 分别应用上述两种方法分离MSCs,比较用两种方法的细胞形态、MSCs细胞得率及以及细胞表面标志的差异.结果 两种方法均能有效培养出骨髓MSCs,且获得的第二代MSCs细胞形态无明显差异,细胞形态均一,为长梭形或三角形.5 ml骨髓细胞分离培养后,Ficoll密度梯度离心法获得的MSCs细胞总数显著小于全骨髓培养法(t=.639,P＜0.01).Ficoll密度梯度离心法与全骨髓培养法获得的第二代MSC8 CD29、CD105、CD105、CD34阳性表达率的差异无统计学意义(t分别为0.809、0.659、0.277、0.191,P均＞0.05),但前者的HLA-DR阳性表达率明显低于后者,差异具有统计学意义(t=2.347,P＜0.05).结论 与Ficoll密度梯度离心法相比较,全骨髓培养法分离MSCs具有培养时间较短,细胞得率较多的优点,虽纯度相对较低,但仍是一种较好的MSCs分离方法.

A comparative study of two culture methods of human bone marrow mesenchymal stem cells

Objective To compare density gradient centrifugation using ficoll and the whole bone marrow method as solutes respectively to separate human mesenchymal stem cells (MSCs)from bone marrow.Methods MSCs were isolated from bone marrow of voluntary normal adults by methods mentioned above respectively. The MSCs were observed in morphology and compared in specific cell-markers after two passages.Results MSCs were isolated from bone marrow of voluntary normal adults by methods mentioned above respectively, were both homogeneous spindle-like or triangle-like cells. The morphology of MSCs gained from two methods had no significant difference. The number of MSCs isolated by density gradient centrifugation was markedly lower than that by the whole bone marrow method (P<0.01).Positive rates of CD29, CD-105, CD166 and CD34 in cells separated by density gradient centrifugation or the whole bone marrow method were similar(P>0.05).But Positive rates of HLA-DR in cells separated by density gradient centrifugation markedly lower than that by the whole bone marrow method(P<0.05).Conclusion As compared to density gradient centrifugation, the whole bone marrow method has a lower MSCs purity and higher number of MSCs.MSCs can be obtained with different methods according to the fact and demand in clinical tissue engineering.