人骨髓间充质干细胞体外分离培养的实验研究

Isolation and Culture of Human Marrow Mesenchymal Stem Cells in Vitro

目的探索体外分离培养人骨髓间充质干细胞的方法并观察基本生长特性,为今后研究其分化提供实验依据。方法:采用Ficoll-Paque淋巴细胞分离液分离人骨髓间充质干细胞,体外培养扩增,在倒置相差显微镜下观察细胞形态变化,细胞计数法绘制生长曲线,流式细胞仪检测细胞周期。结果:密度梯度离心法可获得较高纯度的骨髓间充质干细胞,细胞形态呈圆形、三角形或棒杆状,48h后细胞开始贴壁,4-5d可见有集落形成,2w左右可达到基本融合。生长曲线示骨髓间充质干细胞增殖活跃,流式细胞仪检测显示约有86%的细胞处于G0、G1期。结论:人骨髓间充质干细胞在体外培养条件下生长性状稳定,增殖能力较强,可作为肝组织工程中理想的种子细胞来源。

Objective To explore a practical method of the isolation and culture of human marrow mesenchymal stem cells（hMSCs） in vitro, and to observe their living characteristics , and to offer reference for their differentiation in the future. Methods hMSCs were extracted from human marrow with Ficoll-Paque reagent and proliferated in vitro. The morphology of cells was observed under phase-contrast microscope and the growth curve was drawn by cytometry method. The cell cycle was tested by flow cytometer. Results hMSCs were collected in higher purity by means of gradient centrifugation in Ficoll-Paque. The modalities of cells maybe round, triangle or virgulate, hMSCs began to be adhesive after culture for 48h, and the colony could be formed on day 4 and 5. The basic fusion could be observed in about 2 weeks. The active proliferation of hMSCs was showed by the growth curve. About 86% of MSCs were found at G0,G1 phase by flow cytometry, Conclusion SCs are of steady character and strong proliferation in vitro and can be used as a potential cell source for liver tissue engineering.