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目的:改良大鼠骨髓基质干细胞全骨髓贴壁培养法,并进行Hoechst体外标记初步研究。方法:采用改良全骨髓贴壁法分离扩增大鼠骨髓基质干细胞,以MTT比色实验观察细胞生长状态并绘制生长曲线,行体外诱导向成骨细胞及脂肪细胞分化,以流式细胞术检测细胞表面抗原。以Hoechst体外标记大鼠骨髓基质干细胞,荧光显微镜下计数并观察Hoechest对细胞生长状态的影响。结果:改良全骨髓贴壁培养法能够在较短时间内获得纯化并具备高活性的骨髓基质干细胞,并成功诱导其分化为成骨细胞及脂肪细胞。流式细胞术检测显示细胞表面CD29、CD90、CD44均为阳性,CD45、CD34均为阴性。荧光显微镜观察Hoechst能够在体外成功标记骨髓基质干细胞,并对细胞的生长无明显影响。结论:经改良后的全骨髓贴壁培养法是获得纯化骨髓基质干细胞的一种更为简捷、高效、经济的途径,Hoechst可作为体外标记大鼠骨髓基质干细胞的标记物。
OBJECTIVE: To improve whole bone marrow adherent culture of rat bone marrow stromal stem cells and to study the Hoechst labeling in vitro. Methods: Bone marrow stromal cells (MSCs) were isolated and purified by modified whole bone marrow adherent method. The cell growth status was observed by MTT colorimetric assay and the growth curve was drawn. The osteoblasts and adipocytes were induced to differentiate in vitro by flow cytometry Cell surface antigen. Rat bone marrow stromal cells were labeled with Hoechst in vitro and counted under a fluorescence microscope to observe the effect of Hoechest on cell growth. Results: The modified whole bone marrow adherent culture method can obtain purified and highly active bone marrow stromal cells in a short time and successfully induce osteoblasts and adipocytes differentiation. Flow cytometry showed that CD29, CD90 and CD44 were all positive on the cell surface, while both CD45 and CD34 were negative. Fluorescence microscopy showed that Hoechst successfully labeled bone marrow stromal cells in vitro and had no significant effect on cell growth. Conclusion: The improved whole bone marrow adherent culture method is a simple, efficient and economical way to obtain purified bone marrow stromal cells. Hoechst can be used as a marker for in vitro labeling of rat bone marrow stromal cells.