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目的研究大鼠骨髓间充质干细胞(BMSCs)的体外分离培养;观察Brdu、DAPI以及Hoechst33342体外染色的合适时间和最佳剂量,比较三种方法的优缺点。方法 BMSCs取自SD雄性大鼠的股骨和胫骨,然后采用贴壁分离培养法对BMSCs进行分离培养,当BMSCs培养至第三代时,采用流式细胞仪对其表面抗原CD34、CD44、CD45进行测定;同时分别用Brdu、DAPI以及Hoechst33342对BMSCs进行染色标记,Brdu标记效果用免疫细胞化学方法检测,DAPI和Hoechst33342的标记率用荧光显微镜观测,最后用MTT检测BMSCs的细胞增值率,观察三种不同方法体外染色的合适时间和最佳剂量,同时比较其优缺点。结果流式细胞仪检测发现BMSCs表达CD44阳性,CD34和CD45阴性;BMSCs染色前后其表面标志表达无明显统计学差异(P<0.05);Brdu染色标记BMSCs的最佳浓度和时间是10μmol/L、48小时;DAPI染色标记BMSCs的最佳浓度和时间是20μg/ml、30分钟;Hoechst33342染色标记BMSCs的最佳浓度和时间是5μg/ml、1小时。结论采用粘附贴壁分离培养法能够有效获取高纯度的BMSCs;运用Brdu、DAPI以及Hoechst33342三种方法标记BMSCs效果良好,方法可靠、合理,为研究BMSCs体内追踪打下了基础。
OBJECTIVE: To study the isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs) in vitro and to observe the appropriate time and dosage of Brdu, DAPI and Hoechst33342 staining in vitro. The advantages and disadvantages of the three methods were compared. Methods BMSCs were obtained from the femur and tibia of SD male rats. Then BMSCs were isolated and cultured by adherent culture. When BMSCs were cultured to the third generation, the surface antigens CD34, CD44 and CD45 were analyzed by flow cytometry BMSCs were stained with Brdu, DAPI and Hoechst33342 respectively. Brdu labeling was detected by immunocytochemistry. The labeling rates of DAPI and Hoechst33342 were observed by fluorescence microscopy. The cell proliferation rate of BMSCs was detected by MTT. Different methods of in vitro staining of the right time and the best dose, while comparing their advantages and disadvantages. Results The results of flow cytometry showed that BMSCs were positive for CD44 and negative for CD34 and CD45. There was no significant difference in the expression of BMSCs before and after staining (P <0.05). The best concentration and time of Brdu staining for BMSCs were 10μmol / L, 48 hours. The best concentration and time of DAPI-labeled BMSCs were 20μg / ml for 30 minutes. The best concentration and time of Hoechst33342-stained BMSCs was 5μg / ml for 1 hour. Conclusion BMSCs with high purity can be effectively obtained by adherence adhering wall separation culture method. The BMSCs labeled with Brdu, DAPI and Hoechst33342 are effective, reliable and reasonable, which lays the foundation for the study of BMSCs tracing in vivo.