成人骨髓间充质干细胞中各亚群的比较研究

Comparative Study on Various Subpopulations in Mesenchymal Stem Cells of Adult Bone Marrow

为了观察成人骨髓间充质干细胞在体外培养中的形态 ,比较不同亚群细胞的免疫表型和细胞周期等生物学性状的差异 ,取正常人骨髓单个核细胞 ,进行间充质干细胞 (mesenchymalstemcell,MSC)培养 ,倒置相差显微镜观察MSC不同亚群细胞的形态 ;流式细胞术检测MSC不同亚群细胞的表型并进行细胞周期分析 ;用 10 μm滤膜将不同群体细胞分离 ,分别接种半固体培养体系。结果表明 :①MSC在体外培养中明显分为两群 ,成纤维样梭形细胞为成熟的MSC ,即mMSC ;体积很小的圆形细胞为RS细胞 (rapidlyMSCself renewingcells) ;② 99%的RS细胞处于G0 G1 期 ,mMSC中处于G0 G1 期的细胞占 90 %。③两群细胞的系定向抗原均为阴性 (CD34、CD4 5、CD3、CD19、CD33、HLA DR、CD38等 ) ,而CD90、CD10 5、C16 6、CD2 9、CD4 4、CD4 9e、CD5 4、CD13呈阳性表达 ,但RS细胞表面这些抗原表达的阳性率和平均荧光强度都明显低于mMSC ,而CD117和KDR的表达则显著高于mMSC。④半固体培养 4 - 5周后两种细胞群体均未见造血细胞集落形成。结论 :MSC是一个异质性的细胞群体 ,其中包含的RS细胞可能是一种更原始的中胚层前体细胞 ,具有更强的自我更新和多向分化潜能。

To explore the difference of biological characteristics between two subpopulations of adult bone marrow mesenchymal stem cells (MSC), this study was designed to observe the morphological feature and immunophenotype of the adult MSC in the ex vivo culture, the monouclear cells isolated from normal adult bone marrow were cultured in DMEM with 10% fetal bovine serum. Cell morphology, immunophenotype and cell cycle of two different subgroups were investigated. Cells from 80% confluence were passed through a 10 μm filter, then the fillered cells were cultured in the semisolid methylcellulose medium. The results showed that (1) two different subpopulations were observed in the ex vivo culture. The fibro-like cell was called mature MSC (mMSC) and the smaller round cell was defined rapidly as MSC self-renewing cells (RS cells); (2) the average proportion of cells in G 0/G 1 of RS cells was approximately 99%, but that of mMSCs was 90%; (3) both of the two populations were negative on the lineage-committed antigen (such as CD34,CD45,CD3,CD19,CD33,HLA-DR,CD38), while positive on the expression of CD90,CD105,C166,CD29,CD44,CD49e,CD54,CD13. However, the expression of these antigens on RS cells was weaker than that on mMSC, but CD117 and KDR were higher expressed when compared with the mMSC; (4) after 4 to 5 week semisolid culture, no hematopoietic progenitor cell colonies were observed. It is concluded that adult MSCs are heterogeneous in that distinct morphological populations exist. The RS cells appear to be the more primitive with greater potential for self-renewal and multilineage differentiation.