大鼠骨髓间充质干细胞的分离培养及表型和功能特点

目的 建立体外分离纯化、培养扩增大鼠骨髓间充质干细胞(mesenchymal stem cells，MSCs)的方法，探讨其表型和功能特点。方法 用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓MSCs，传代扩增，测定生长曲线，通过形态学观察，细胞化学及免疫细胞化学法分析大鼠骨髓MSCs的表型和功能特点。结果 MSCs属骨髓中单个核细胞，密度梯度离心结合贴壁培养法能有效分离纯化大鼠骨髓MSCs，在含10％小牛血清的L-DMEM中，1、3、5代细胞生长曲线基本相同，增殖快，每传代一次细胞约增加2．2倍。细胞呈均一的成纤维细胞样，表达CD44、CD54、纤维粘连蛋白(fibronectin，FN)、Ⅰ型胶原(collagenⅠ)。加入地塞米松(Dex 10^-8mol／L)、β-甘油磷酸钠(β-GP 10mmol／L)、抗坏血酸(AA50μg／ml)可诱导MSCs分化为成骨细胞，碱性磷酸酶(alkaline phosphatase，ALP)活性增高，形成矿化结节。结论 所分离、培养的细胞具有间充质干细胞的表型和功能特点，建立了体外分离纯化、培养扩增大鼠骨髓MSCs的方法。

Isolating and Culturing Rat Marrow Mesenchymal Stem Cells and Studying Their Phenotypical and Functional Properties

OBJECTIVE: To establish a method for isolating and cultivating mesenchymal stem cells (MSCs) from SD rat bone marrow and to study their phenotypical and functional properties. METHODS: MSCs from rat bone marrow were separated and purified by gradient centrifugation and adherence to the culture plastic; then the cells were expanded by subculture successively. The growth curves were drawn, and the morphology was observed. In an attempt to analyze immuno- and adhesive-phenotype and differentiation properties, the MSCs were evaluated with cytochemical and immunocytochemical methods. RESULTS: MSCs belonged to the mononuclear cells of marrow, they could be isolated and purified by gradient centrifugation and adherence to the culture plastic; their living behavior was quite stable in Dulbecco's Modified Eagle's Medium with low glucose (L-DMEM) containing 100 ml/L newborn bovine serum; the growth curves of passage 1, 3 and 5 were much similar, exhibiting a 2.2-fold increase in cell number after each passage. The cells were noted to have a large expansive potential and a typical fibroblast-like morphology, and they uniformly expressed CD44, CD54, fibronectin and collagen I. When incubated in medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, the MSCs underwent differentiation into osteoblasts, showing positive stain of alkaline phosphatase activity and mineralized nodules. CONCLUSION: The cells obtained in this experiment possess phenotypical and functional properties of mesenchymal stem cells, and the method for isolating and culturing rat marrow-derived MSCs has been established.