体外培养人骨髓间充质干细胞的生物学特性

The biological characteristics of mesenchymal stem cells from human bone marrow

目的 体外分离培养人骨髓间充质干细胞 (MSCs)并研究其一般的生物学特性。方法 通过密度梯度离心法分离MSCs、体外培养传代细胞计数、流式细胞仪检测细胞增殖周期、表面分子测定以及细胞化学染色了解人MSCs一般的特点。结果 人MSCs经过体外培养均一地表达CD2 9、CD4 4、CD16 6 ,而CD34、CD4 5阴性。细胞化学结果显示 ,细胞糖原 (PAS)强阳性 ,脂肪 (SB)为阴性 ,5 %～ 10 %细胞碱性磷酸酶 (ALP)阳性。细胞周期分析表明 :G0 /G1、S和G2 /M所占比例分别为 83 11%、5 17%和 11 72 %。分离细胞在成骨诱导体系下进一步向成骨细胞分化。结论 密度梯度离心法能够分离纯化MSCs有其特殊的生物学特点 ,能够向成骨细胞分化。

Objective The aim is to know the biological characteristics of mesenchymal stem cells (MSCs) from human bone marrow. Methods MSCs were isolated by density gradient centrifugation with percoll at a density of 1 073 g/L in order to eliminate unwanted cell types. The mononuclear cells were incubated in low glucose dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum. MSCs were highly purified and culture-expanded. The morphology,phenotype,histology and biological properties of the cultured MSCs were investigated. Results MSCs possessed some unique phenotypes. MSCs were fusiform shape,and the growth speed was swiffed in the earlier period. The amplification speed of cells became slower after 10 generations. Flowcytometry analysis showed that MSCs were positive for CD29,CD44,CD166,and negative for CD34,CD45. Cytochemistry staining evaluation revealed that MSCs were homogeneously positive for glycogen (periodic acid Schiff reaction,PAS),negative for Sudan black reaction (SB) and around 5% of them were positive for alkaline phosphatase (ALP). Cell cycle studies showed there were 83.11%,5.17% and 11.72% percentage of the cells in G_0/G_1,S and G_2/M respectively. Conclusion The MSCs have peculiar biological characteristics of stem cells which differ the cells from clusters of HSCs.