Objective To observe the labeling conditions of rat BMSCs using Hoechst33342, BrdU, and DAPI, respectively, and to compare the effect of each method. To observe cell growth and the fluorescence duration by culturing mouse BMSCs of transgenic green fluorescence mice, and to compared the superiority between mouse BMSCs and rat stem cells labeled with fluorescent. Methods Rat BMSCs were extracted using adherence screening, and cultured to the 3rd generation. The surface antigen was detected using flow cytometry. Staining was performed after osteogenic or adipogenic induction. Rat BMSCs were labeled with Hoechst 33342, BrdU, or DAPI, respectively. The proliferation of labeled rat BMSCs was detected. The fluorescence duration was observed under microscopy. Cell morphology and fluorescence duration of mouse BMSCs, which were cultured for different time, were observed. The advantages and disadvantages were compared with those of the 3 labeling methods mentioned above. Results Hoechst 33342, BrdU, and DAPI were available for BMSCs labeling. The labeling process of BrdU was long. None fluorescence quenching existed in the pre-labeled. Cells with positive labeling presented after the process of fluorescent immunohistochemistry. The labeling process of Hoechst 33342 and DAPI was short. The fluorescence duration was long. Because light should be avoided during the whole process, this brought some difficulties to the operation. No effect of the three methods on cell proliferation rate had been observed. The growth of BMSCs of transgenic mice was much slower than that of rat BMSCs. Its advantages were free of labeling and long fluorescence duration. So it was more convenient and feasible for cell tracking study. Conclusion Stem cells have the proficiency of multiple differentiations. They have great value of research and application. Stem cell labeling can provide a better platform for the mechanism study of stem cell migration and homing.%目的 观察Hoechest33342、BrdU、DAPI对大鼠BMSCs标记情况,比较其标记效果的差异性;培养转基因绿色荧光小鼠的BMSCs,观察细胞生长情况和其荧光持续时间,比较其与荧光标记后的干细胞之间的优越性.方法 贴壁筛选提取大鼠BMSCs,培养至第三代,流式细胞仪检测其表面抗原,成骨、成脂诱导后染色;Hoechest33342、BrdU、DAPI标记大鼠BMSCs,检测标记后的大鼠BMSCs增殖率,显微镜下观察其荧光持续时间;观察转基因绿色荧光小鼠BMSCs在培养的不同时间段的细胞形态、荧光持续时间以及同上边3种标记方法比较的优缺点.结果 Hoechest33342、BrdU、DAPI可用于对BMSCs的标记;BrdU标记过程较长,在前期不存在荧光猝灭缺点,标记后经过荧光免疫组化处理后可见阳性细胞,Hoechest33342、DAPI标记过程较短,荧光持续时间较长,但此过程中必须避光,对操作带来了一定的困难.3种方法标记后对细胞增殖率无影响;转基因小鼠BMSCs生长较大鼠干细胞生长较慢,其优点是不用标记且荧光持续时间较长,用于细胞追踪研究更为方便可行.结论 干细胞有多向分化的潜能,因此有很好的研究价值和应用价值,对干细胞进行标记,为干细胞迁移、归巢机制的研究提供更好的研究平台.
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