人重组Fas配体体外诱导细胞周期特异性凋亡模型的建立

Establishment of Cell Cycle Specific Apoptosis Model Induced by Recombinant Human Fas Ligand in vitro

目的运用人重组Fas配体(rhFasL),体外诱导稳定而又典型的Fas介导的细胞周期特异性凋亡模型,为针对其细胞凋亡过程中的细胞周期机制研究、肿瘤的生物学治疗和自身免疫性疾病的防治等提供科学的研究平台。方法以白血病细胞系(Molt-4和Jurkat细胞系)和进入细胞周期的外周血淋巴细胞为载体,用人重组Fas配体诱导细胞6～36h后,用亚G1峰法、传统的AnnexinⅤ/PI和改良后的API法进行标记并用流式细胞术检测细胞凋亡情况。结果改良后的API法显示人重组Fas配体诱导的细胞凋亡具有细胞周期特异性及始动位点;传统的AnnexinⅤ/PI显示在将细胞凋亡控制在10%～20%并以早期细胞凋亡为主时诱导的模型最典型;亚G1峰法只能检测晚期细胞凋亡和进行DNA直方图分析。结论人重组Fas配体体外诱导的细胞凋亡具有细胞周期特异性并始动于G1期,联合应用传统的Annex-in-Ⅴ/PI及改良后的API法能为建立稳定而又典型的体外Fas介导的细胞周期特异性凋亡模型提供技术支撑。

Objective To establish a stable cell cycle specific model induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods After leukaemia cell lines and peripheral blood lymphocytes which entered cell cycle progression were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1 , traditional Annexin-V/PI and improved API methods and analyzed by flow cytometry. Results The improved API method demonstrated the cell cycle specificity and initiation site of apoptosis induced by recombinant human Fas ligand. The traditional AnnexinV/PI method showed the most appropriate condition for the establishment of typical cell cycle-specific model. The sub-G1 method could only detect late apoptosis and DNA histogram. Conclusion Fas-mediated apoptosis was cell cycle-specific and initiated at G1 phase. Based on the improved API and traditional Annexin V/PI methods, the establishment of stable and typical cell cycle-specific model in Fasmediated apoptosis in vitro was feasible.