人骨髓间充质干细胞的扩增培养及向成软骨样细胞的定向诱导分化

Expansion and chondrogenic induction of human bone marrow mesenchymal stem cells

从人骨髓中分离和培养间充质干细胞(MSCs),在原代和传代培养中其形态学上均为贴壁、纤维状的细胞,随着代次的增加细胞形态更加趋于一致.流式细胞仪鉴定CD45、CD34、CD117、HLA-DR阴性,CD29、CD166阳性,并随着培养代次的增加,阳性比率和阴性比率出现相应的上升和降低,说明细胞中MSCs纯度增加.第三代细胞大约有80%处于G0/G1期,S+G2+M期约20%,说明MSCs有强大的增殖潜能.在传代培养中的最适接种密度为5000个/cm2左右.MSCs在体外经历了滞缓期、对数生长期、平稳期3个时期,传代培养的MSCs生长速度比原代的MSCs明显快很多.采用程序降温法对MSCs进行冻存,60～90d后对冻存的MSCs进行复苏,发现MSCs仍可在体外良好生长4～6代,特定的MSCs样本可以扩增10代,说明经过冻存的细胞仍具有良好的增殖能力.对4例标本的前三代MSCs在体外利用TGF-β1和维生素C诱导两周后,用RT-PCR证明MSCs表达软骨特异性的II型前胶原的mRNA,说明培养的细胞具有向成软骨样细胞分化的潜能,由此进一步证实其为MSCs.

MSCs from human BM were isolated by Ficoll density centrifugation cultured and expanded in DMEM-LG containing FBS. MSCs are adherent and fibroblastic, and maintained similar morphology as passaging. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD166, and negative for CD34, CD45, CD117, and HLA-DR. At passage 3, cell cycle status analysis by measuring DNA content revealed that a small population of cells was engaged in proliferation (S^G-2~M=20), and more than 80 of cells were in G-0/G-1 phase. The number of cells in G-0/G-1 phase decreased as passaging. In passage culture, it is proper for the cells to be plated at the density of 5 000 cells/cm^2. MSCs in passages 1~3 were frozen in liquid nitrogen by two-step frozen procedure. After 60～90 days, the frozen MSCs were thawed and then proliferated in vitro for 4～6 passages,10 passages especially for sample 17. It was suggested that the frozen MSCs have a high expansion ability. In passages 1~3, four cases were induced in vitro with the combination of TGF-β-1 and Vitamine C for two weeks and expressed articular cartilage matrix-procollagen II mRNA.