人骨髓间充质干细胞的扩增及向成软骨样细胞的诱导分化
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，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，并随着培养的MSCs代次的增加，阳性比率和阴性比率出现相应的上升和降低，说明细胞纯度增加。第三代的MSCs大约有80%处于G0/G1期，S+G2+M期约20%左右，说明MSCs有强大的增殖潜能。MSCs在传代培养中的最适接种密度约为5.0×103/cm2 左右。MSCs在体外经历了滞缓期、对数生长期、平稳期三个时期，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，60～90天后对冻存的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。对四例标本的前三代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，说明我们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 and cultured expanded in DMEM-LG containing FBS. MSCs are adherent and fibroblastic, and maintained similar morphology with 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 the cells was engaged in proliferation (S+G2+M=20%), and more than 80% of cells were in G0/G1 phase. The number of cells in G0/G1 phase decreased with passage. In passage culture, It is proper for the cells plated at the density of 5000 cells/cm2 to propagate. The MSCs of passage 1 to 3 were froze in liquid nitrogen by two-step frozen procedure ,after 60～90 days, we thawed the frozen MSCs and found the cells can proliferate about 4～6 passages in vitro，10 passages especially for sample 17. It suggested that the frozen MSCs still have good expansion ability
邱丽燕、王金福
细胞生物学分子生物学生物工程学
骨髓间充质干细胞 扩增 成软骨样细胞
Mesenchymal stem cells (MSCs) expansion chondrocyte
邱丽燕,王金福.人骨髓间充质干细胞的扩增及向成软骨样细胞的诱导分化[EB/OL].(2004-01-14)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/200401-57.点此复制
评论