大鼠骨髓间充质干细胞体外培养及CM-DiI标记的实验研究

摘要

目的 建立骨髓间充质干细胞(MSCs)的体外分离纯化、扩增以及标记的方法,为后期体内实验提供依据.方法 采用全骨髓培养和差速贴壁法分离纯化MSCs,培养期间观察细胞形态,流式细胞仪检测细胞周期和表面标记物,成脂成骨分化诱导鉴定.MSCs经氯甲基苯甲酰氨荧光染料(CM-DiI)标记后,荧光显微镜观察荧光标记情况,流式细胞仪检测标记率,并通过绘制生长曲线明确CM-DiI对MSCs生长增殖的影响.结果 MSCs接种24~48 h后,贴壁细胞多为圆形、多角形、梭形,传至第3代时形态呈均一的长梭形.第3代MSCs高表达CD44,低表达CD106,不表达CD11b、CD45,85%以上细胞处于G0/G1期;成脂成骨诱导分别经油红O和茜素红染色均呈阳性.CM-DiI标记后荧光显微镜下细胞呈红色荧光,体外培养2周荧光未见明显减弱;流式细胞术检测显示标记率达95%以上,标记后细胞生长增殖不受影响.结论 通过全骨髓贴壁法及其扩增后可获得大量纯度较高的间充质干细胞,CM-DiI标记是一种简单、稳定、无毒的细胞标记方法.%Objective To establish a method for isolation, purification, proliferation and labeling of bone marrow mwsenehymal stem cells ( MSCs )in vitro. Methods MSCs were isolated and purified by whole bone marrow adherence and differential adhesion separation. The morphological characteristics were observed in incubation period. Cell cycle and surface makers were analyzed by flow cytometry as well as induction of adipogenic and osteogenic differentiation for identification of MSCs. MSCs were labeled with chloromethyl-benzamidodialkylarbocyanine( CM-DiI), observed by fluorescence microscope and analyzed by flow cytometry. The impact of CM-DiI on growth and proliferation of MSCs were recorded by drawing growth curve. Results MSCs mostly showed circular, polygonal, spindle after inoculation for 24 ~48 h. At the third passage, cells formed a uniform long spindle shape. MSCs at passage 3 were strongly positive for CD44, weakly positive for CD106, but negative for CD11b and CD45. There were more than 85% of MSCs in the G0/G1 phase. After adipogenic and osteogenic induction, oil red O and alizarin red staining were positive respectively. CM-Dil labeled cells showed red fluorescence under fluorescence microscope. Florescence emerged by CM-DiI was not significantly weakened after labeled 2 weeks in vitro. Cell labeling rate was up to 95% . CM-DiI did not effect on cell growth and proliferation. Conclusion A great number of MSCs with high purity have been obtained by method of whole bone marrow adherence and amplification. The tracing method of CM-DiI labeling cells is simple, stable, and non-toxic.