晚期糖基化终产物修饰人血清白蛋白对人血管内皮细胞的生长抑制作用

目的　探讨晚期糖基化终产物 (AGE)在糖尿病难愈创面形成和发展中的作用机制。方法　将人脐静脉内皮细胞株ECV30 4细胞与不同浓度的AGE修饰人血清白蛋白 (AGE HSA)在体外共同培养。应用四甲基偶氮唑蓝 (MTT)比色法、锥虫蓝排斥实验和活细胞计数检测AGE HSA对血管内皮细胞的生长抑制作用。采用AnnexinVFitc和碘化丙啶 (PI)染色 ,通过流式细胞仪检测细胞凋亡百分率 ,同时应用荧光共聚焦显微镜观察凋亡或死亡细胞AnnexinVFitc和PI的荧光染色 ;利用透射电镜和光镜观察AGE HSA对内皮细胞的形态学影响。结果　内皮细胞经 12 .5、2 5和 5 0 μg/mlAGE HSA干预 2d后 ,各干预组细胞数与对照组比较无明显差异 (均P >0 0 5 ) ,而于 4d和 6d后明显低于对照组 ,细胞增殖抑制率显著上升。内皮细胞经 10 0或 2 0 0 μg/mlAGE HSA干预 6、12、2 4、4 8h后 ,MTT法测其吸光度A值在各时相点均显著低于对照组 (P <0 0 1) ,同时内皮细胞出现特异性的凋亡形态学变化 ,AnnexinVFitc阳性和 /或PI阳性的细胞逐渐增多 ,流式细胞仪测定的各时相点凋亡细胞百分率均显著高于对照组 (P <0 0 1) ,且凋亡或死亡细胞数量随AGE HSA作用时间及浓度的增加而增多。结论　AGE HSA能抑制内皮细胞增殖 ,并可以诱导其凋亡 ,而且与作用时间及浓

Proliferation-inhibiting effect of advanced glycation end products modified human serum albumin to vascular endothelial cell ECV304

OBJECTIVE: To study the proliferation-inhibiting and apoptosis-inducing effects of advanced glycation end products (AGE) modified human serum albumin (AGE-HSA) on human vein endothelial cells. METHODS: Human umbilical vein endothelial cells ECV304 were cultured in vitro with AGE-HSA of the concentrations of 12.5, 25, 50, 100, and 200 micro g/ml for 6, 12, 24, or 48 hour, then 20 micro l of 5 mg/ml MTT were added and the optical density (OD) at each time point was determined. Another ECV304 cells were cultured with AGE-HAS for 2, 4, or 8 days and then were stained with trypan blue to calculate the number of dead cells so as to calculate the proliferation-inhibiting rate. Another ECV304 cells were cultured with AGE-HAS for 6, 12, 24, or 48 hours and then stained with annexin V Fitc and propidium iodide (PI). Flow cytometry was used to calculate the annexin V Fitc positive cells (early and middle stage apoptotic cells) and Annexin V Fitc/PL positive cells (late apoptotic cells). Inverted microscope, transmission electron microscope, and fluorescence microscope were used to observe the histological changes of apoptotic cells. FCV304 cells incubated with HSA of the above-mentioned and without addition of the other agents concentrations were used as controls. RESULTS: The OD values of ECV304 cells cultured for 48 h with low concentrations (12.5, 25, and 50 micro g/ml) of AGE-HSA were not significantly different from those of the control (1.104 +/- 0.080, 1.098 +/- 0.097 and 1.059 +/- 0.122 VS. 1.159 +/- 0.088, all P > 0.05). The OD values of ECV304 cells cultured with low concentrations of AGE-HSA for 4 days and 6 days were significantly lower than those in the control group. The OD values of ECV304 cells cultured with high concentrations (100 and 200 micro g/ml) of AGE-HSA for 6 - 48 hours decreased to 0.117 +/- 0.033 and 0.081 +/- 0.020 in comparison with that of the control group (P < 0.01). Flow cytometry and fluorescence microscopy showed higher proportions of apoptotic cells among the ECV304 cells cultured with high concentrations of AGE-HAS than among the control cells at each time point (P < 0.01). The numbers of cells in the control group exponentially increased after culture for 2, 4, and 6 days. The number of cells cultured with low concentrations of AGE-HAS for 2 days was not significantly different from that of the control group (P > 0.05), however, the numbers of cells cultured with low concentrations of AGE-HAS for 4 and 6 days were significantly lower than those of the control group (both P < 0.01). The numbers of cells cultured with 100 or 200 micro g/ml AGE-HAS for 2 days were significantly lower than those of the control group (both P < 0.01) with a proliferation-inhibiting rate of 39.56% +/- 2.82% and 60.32% +/- 4.51% respectively. The apoptotic rates in cells cultured with low concentrations of AGE-HAS for 48 hours were not significantly different from those in the control group. The apoptotic rates in cells cultured with 100 or 200 micro g/ml AGE-HAS for 6, 12, 24, or 48 hours were significantly higher than those in the control group (all P < 0.01). The apoptotic rates in 200 micro g/ml group at different time points were significantly higher than those in the 100 micro g/ml group (P < 0.05 or 0.01). The apoptotic rate and number of apoptotic cells increased along with the increase of culture time and concentration of AGE-HAS. Microscopy showed morphological changes among the cells cultured with 100 micro g/ml AGE-HAS for 6, 12, 24, and 48 hours and the numbers of apoptotic cells, mainly late apoptotic cells, and dead cells increased remarkably since the cells were cultured for 48 hours. CONCLUSION: AGE-HSA inhibits the proliferation of vascular endothelial cells and induces apoptosis in dose and time dependent manner. AGE modification-induced pathobiological cascade may be involved in the pathogenesis of impaired wound healing in diabetes by the mechanism of angiogenesis retardation.