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目的 :建立结合珠蛋白 (Hp) DNA双参数流式细胞技术 ,以探讨放射损伤小鼠骨髓细胞中Hp含量与细胞周期和凋亡的关系。方法 :小鼠骨髓细胞经 70 %乙醇固定后 ,Triton_X 10 0破膜 ,间接免疫荧光技术标记Hp ,PI标记DNA ,上流式细胞仪测试。用ModFit软件设双门法分析Hp+ 和Hp_细胞的细胞周期和凋亡 ,CELLQuest软件分析Hp+ 细胞比例。结果 :γ射线照射后 6h小鼠骨髓有核细胞中Hp+ 细胞的比例明显高于未照射对照组 ,骨髓细胞的凋亡来自于Hp_细胞 ,Hp+ 细胞未见凋亡 ,Hp_细胞发生的G2 M阻滞比Hp+ 细胞严重。这些信息是单用Western印迹法或单纯DNA分析所不能提示的。结论 :Hp DNA双参数流式细胞技术可用于分析Hp与细胞周期和凋亡的关系 ,是一种快速、客观的分析方法。
OBJECTIVE: To establish a double-parameter flow cytometry (Hp) DNA technique to investigate the relationship between Hp and cell cycle and apoptosis in bone marrow cells of irradiated mice. Methods: The bone marrow cells were fixed with 70% ethanol, Triton_X 10 0 rupture membrane, indirect immunofluorescence technique labeled with Hp, PI-labeled DNA, and tested by flow cytometry. The ModFit software was used to analyze the cell cycle and apoptosis of Hp + and Hp_ cells by the double-gate method. CELLQuest software was used to analyze the proportion of Hp + cells. Results: The ratio of Hp + cells in bone marrow nuclei of mice at 6h after γ-ray irradiation was significantly higher than that of non-irradiated control group. The apoptosis of bone marrow cells was from Hp_ cells, no apoptosis was observed in Hp + cells and G2 M block more severe than Hp + cells. This information can not be prompted by Western blot or DNA analysis alone. Conclusion: Hp DNA dual-parameter flow cytometry is a rapid and objective method for analyzing the relationship between Hp and cell cycle and apoptosis.