羟自由基、低密度脂蛋白和胆固醇对血管内皮细胞钙响应能力的影响

Effects of hydroxyl radicals,low density lipoprotein and cholesterol on the calcium-responding behavior of vascular endothelial cells

目的 :探讨分别经羟自由基、低密度脂蛋白和胆固醇处理后 ,血管内皮细胞对外钙浓度增加的响应能力的改变 ,为研究动脉粥样硬化的发生发展机制提供新线索。方法 :用人脐静脉内皮细胞作为模型细胞 ,分别用羟自由基、低密度脂蛋白、胆固醇和氧化胆固醇进行单独或组合处理 ,利用Fluo 3荧光标记细胞内游离Ca2 + ,以激光共聚焦手段考察了当细胞外液 [Ca2 + ]增加时胞内 [Ca2 + ]的变化。结果 :正常血管内皮细胞对胞外 [Ca2 + ]增加响应灵敏 ,胞内 [Ca2 + ]升高到一定程度后 ,迅速下降至初始水平 ;细胞经羟自由基处理后 ,对Ca2 + 的响应明显迟钝 ,胞内[Ca2 + ]达到峰值的时间延长了 35 0s ,细胞形态也有损伤迹象 ;细胞经低密度脂蛋白处理后 ,胞内 [Ca2 + ]达到峰值的时间延长了 2 5 0s,且对 [Ca2 + ]的调节作用明显失常 ,使细胞处于高钙水平 ;而细胞经羟自由基和低密度脂蛋白依次处理后 ,细胞内 [Ca2 + ]达到峰值的时间延长了 5 0 0s,损伤程度更大 ,当胞外 [Ca2 + ]增加时细胞破裂死亡 ;细胞经胆固醇处理后 ,当胞外 [Ca2 + ]突然升高时 ,胞内 [Ca2 + ]不仅没有升高 ,反而缓慢降低 ;细胞经羟自由基和胆固醇依次处理后 ,对胞外 [Ca2 + ]的突然升高反应紊乱。结论 :羟自由基。

Objective:To investigate the effects of hydroxyl radicals, low density lipoprotein and cholesterol on the response of vascular endothelial cells to the change in extracellular calcium ion concentration. Methods:After treatment with the above mentioned atherogenic factors respectively, the cells were loaded with Fluo 3 and then the response of the cells to the increase in extracellular [Ca 2+ ] was observed under a confocal laser scanning microscope. Results:Normal vascular endothelial cells were sensitive to the increase in extracellular [Ca 2+ ]. The free calcium ions in cytosol increased pulsively until a maximum was reached, and then was reduced to the initial level rapidly. Treatment with hydroxyl radicals produced by Fe 2+ EDTA made the cells rather slow to the change in extracellular [Ca 2+ ]. A morphological examination showed that some damage to the cells was caused. After cultivation with low density lipoprotein (0.10 g·L -1 ), the cells lost most of their capability to modulate intracellular calcium ion and thus the calcium level remained high at the end of the experiment. If a hydroxyl radical treatment preceded the incubation with low density lipoprotein, a more serious damage was observed. Some of the cells were caused to death while the extracellular [Ca 2+ ] increased. Upon exposing to a higher level of extracellular calcium ion, the cytosol [Ca 2+ ] did not increase; contrarily, it decreased gradually. The hydroxyl radical oxidation followed by cholesterol treatment made the cells respond disorderly to the increase in extracellular [Ca 2+ ], indicating that rather serious damage had been caused to the cells. Conclusion:These findings indicate that hydroxyl radicals, low density lipoprotein and cholesterol interfere with the response of vascular endothelial cells to extracellular calcium ions. This may be one of the ways these factors contribute to the initiation and development of atherosclerosis.