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_图文.docVIP

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 邱丽燕,王金福 (浙江大学生命科学学院 浙江杭州 310012 摘要 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G 0/G 1期,S+G 2+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/cm 2 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 。 关键词 骨髓间充质干细胞 扩增 成软骨样细胞 Expansion and Chondrogenic Induction of Human Bone Marrow Mesenchymal Stem Cells Qiu Liyan, Wang Jinfu (College of Life Science, Zhejiang University, Hangzhou, Zhejiang 310012 Abstract 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+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 with passage. In passage culture, It is proper for the cells plated at the density of 5000 cells/cm 2 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. At passage 1 to 3, four cases was induced by the combination of TGF-β1 and Vitamine C in vitro for two weeks and expressed articular cartilage matrix-procollagen II mRNA. Key word Mesenchymal stem cells (M