凋亡细胞线粒体膜电势与物理参数变化的可视化研究

A visualization study on mitochondrial membrane potential and physical parameters of apoptotic cell

目的:通过放线菌酮(CHX)诱导HL-60细胞凋亡模型,探讨线粒体膜电势与细胞物理参数的变化。方法:建立CHX诱导HL-60细胞凋亡模型。于6和18 h时点,利用Annexin V-FITC/PI双染流式细胞术检测凋亡和晚期凋亡细胞及坏死细胞比率;利用JC-1染色流式细胞术,通过对对照组和CHX组FL1/FL2散点图上具有不同荧光强度的细胞群划分为R2,R3,R4 3个细胞群,并分别对这些细胞群在FSC/SSC二维散点图上进行反向设门,分析细胞凋亡过程中线粒体膜电势变化与细胞物理参数间的关系。通过透射电镜观察凋亡细胞形态结构改变。结果:CHX可引起HL-60细胞凋亡;与对照组相比,CHX组R2和R3细胞群物理参数为FSC和SSC均显著增高,但线粒体膜电势无显著性差异,P>0.05;R4细胞群则为FSC降低,SSC增高,P<0.05,且线粒体膜电势降低。CHX可诱导HL-60细胞体积变小,皱缩,并出现凋亡特征,如核固缩形成新月体和质膜"出芽"现象。结论:流式细胞仪FSC/SSC图可视为细胞指纹图谱。本模型中线粒体膜电势降低的细胞变小,且颗粒程度增加,可能与细胞凋亡过程中的结构改变有关。

Aim：To study the relevancy between the cell mitochondrial membrane potential and physical parameters change of apoptotic cell though the cycloheximide （CHX） -induced HL- 60 cell apoptosis model. Methods： Detecting the apoptotic cell ratios by ArmexinV-FITC/PI double stainings flow cytometry at 6 h and 18 h time points. Dividing different regions of JC-1 staining cell according to the fluorescence intensity on the FL1/FL2 dot-plot picture, analyzing the relationship between mitochondrial membrane potential and physical parameters of cells by gate backtrack respectively in the FSC/SSC dot-plot picture. Observe the changes in morphology of the CHX induced apoptotic HL-60 cells by transmission electronic microscope. Results： CHX can induce the cell apoptosis. Compared with the same region of control group, the FSC and SSC fluorescence intensity of R2 and R3 region in CHX group increased significantly, while the mitochondrial membrane potential in these region retain at the same level; the FSCfluorescence intensity decreased significantly in R4 region of CHX group with lower mitochondrial membrane potential, while the SSC fluorescence intensity increased significantly. The CHX treated HL-60 cell showed the typical apoptotie characters, including the condensation of cytoplasm, shrinkage or loss of cell volume and plasma membrane budding. Conclusion：The FSC/SSC dot-plot picture of eytometry is a fingerprint. In the eyeloheximide（CHX）-induced HL-60 cell apoptosis model：The cells with lower mitoehondrial membrane potential became smaller and the density of granules inside increased. This change probably related to the cell uhrastrueture alterations during the apoptofie process.