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目的:探讨体外培养人骨髓基质干细胞(BMSC)的方法和初步诱导BMSC向神经细胞方向分化的方法。方法:采用正常成人献髓者骨髓,分离扩增BMSC,原代培养后将传1～4代细胞按1×104/ml种于24孔板,绘制生长曲线、贴壁率,观察细胞生长及不同浓度的碱性成纤维生长因子(bFGF)对BMSC的作用。以全反式维甲酸(RA)和bFGF为诱导剂,观察诱导前后BMSC的变化。结果:原代BMSC生长状态良好,传至第5代仍保持干细胞特性,bFGF可明显促进BMSC增殖,且呈剂量依赖关系。RA和bFGF诱导12h,BMSC逐渐向神经样细胞转化,胞体收缩成锥形、三角形或不规则形,细胞伸出细长突起,免疫细胞化学鉴定呈Nestin阴性,NSE阳性,GFAP阳性,且NSE阳性细胞数较GFAP阳性为少。结论:BMSC可在体外稳定扩增,且能保持干细胞特性,RA和bFGF可诱导其分化为神经细胞。
Objective: To investigate the method of culturing human bone marrow stromal stem cells (BMSCs) in vitro and the method of inducing BMSCs to differentiate into neurons. Methods: BMSCs were isolated from normal adult donors and cultured. Primary cultured cells were seeded into 24-well plates at 1 × 104 / ml for 1 × 4 cells. The growth curve and cell attachment rate were observed. Effects of different concentrations of basic fibroblast growth factor (bFGF) on BMSCs. All-trans retinoic acid (RA) and bFGF were used as inducers to observe the changes of BMSC before and after induction. Results: The primary BMSCs grew well and maintained the 5th generation of stem cells. The bFGF could promote the proliferation of BMSCs in a dose-dependent manner. After induced by RA and bFGF for 12h, BMSC gradually transformed into neuron-like cells, and the cell bodies contracted into a cone, triangle or irregular shape. The cells extended slender protrusions. The immunocytochemistry showed Nestin-negative, NSE-positive, GFAP-positive and NSE-positive Cells are less positive than GFAP. Conclusion: BMSC can stably proliferate in vitro and maintain the stem cell characteristics. RA and bFGF can induce the differentiation into nerve cells.