血管内皮细胞在传代中的衰老与线粒体膜电位的变化

目的探讨血管内皮细胞在传代过程中的衰老过程及机制。方法新鲜人脐静脉内皮细胞体外培养,免疫组织化学鉴定;电镜观察细胞结构;流式细胞术检测传代细胞周期;特异染料法检测细胞线粒体膜电位,流式细胞仪分析。结果在传代过程中,细胞结构逐渐退化。随着传代次数的增多,细胞从DNA合成前期进入DNA合成期的比例逐渐降低,从DNA合成后期进入分裂期的比例逐渐增加。其中,处于静止期和DNA合成前期的细胞由第2代的74.17%增加至第8代的88.80%,处于DNA合成期的细胞由第2代的20.04%降低至第8代的7.95%。细胞增殖指数由第2代细胞的25.83%降低至第8代细胞的11.41%。代表线粒体膜电位的平均荧光强度逐步降低。结论内皮细胞传代过程中,细胞结构退化,细胞生长渐趋停滞,出现衰老特征。线粒体膜电位降低,线粒体功能下降,可能是细胞衰老的机制之一。

Phenomenon of aging during passage of vascular endothelial cells and its relationship with fluctuation of mitochondrial membrane potential

Objective To study the process of aging and its potential mechanisms during passage of vascular endothelial cells.Methods Human umbilical vein endothelial cells(HUVECs) were cultured in vitro.The changes of HUVECs forms were tracked.Flow cytometry was used to examine the cell cycles and the mitochondrial membrane potential was examined with specific staining.Results In the course of passage,the cellular structures degenerated gradually.With increase in number of passages,the proportion of cells proceeding from DNA presynthesis stage to DNA synthesis stage decreased gradually,while the proportion of cells proceeding from late stage of DNA synthesis to division stage increased gradually.Meanwhile,The cells at resting stage and DNA presynthesis stage increased from 74.17% in second generation to 88.80% in eighth generation and cells at DNA synthesis stage decreased from 20.4% in second generation to 7.95% in eighth generation.The proliferation indices obviously decreased from 25.83%(second generation) to 11.41%(eighth generation,P<(0.01)).The average fluorescence intensity which represented the mitochondrial membrane potential also decreased gradually with increase in number of passages.Conclusions The cellular structures degenerate gradually and cell growth is prone to stagnate at S phase in cells after repeated passages.The drop of mitochondrial membrane potential and damage of mitochondria function are probably the mechanism of senescence of VECs.