同型半胱氨酸对血管内皮细胞表达血栓调节蛋白的影响

Effects of homocysteine on the expression of thrombomodulin in human endothelial ECV 304 cells

目的 :观察不同浓度的同型半胱氨酸对血管内皮细胞表达血栓调节蛋白的影响。方法 :传代培养ECV 30 4细胞 ,随机给予 7种不同浓度的Hcy(0 μmol/L、2 5 μmol/L、5 0 μmol/L、10 0 μmol/L、1μmol/L、5 μmol/L、10mmol/L) ,处理 2 4h后 ,检测细胞活力和上层培养液中的LDH含量 ;用ELISA方法检测细胞上层培养液中的可溶性血栓调节蛋白 (sTM )的浓度和细胞总的TM含量。结果 :1mmol/L以下的Hcy对细胞的活力 ,培养液中的LDH含量和sTM浓度无影响。 5mmol/L的Hcy使细胞的活力开始下降 ,10mmol/L的Hcy还可以导致细胞LDH漏出量的增加 ,sTM也相应升高。不同处理组均可见TM的表达。超过 1mmol/L的Hcy作用 2 4h可以使细胞总的TM含量显著增加。结论 :生理浓度的Hcy不直接对内皮细胞造成影响 ,但超生理浓度的Hcy可以使TM的合成增加。

Objective:To evaluate the effects of homocysteine on the expression of thrombomodulin in cultured endothelial cells. Methods:Human umbilical veins endothelial cells (ECV-304 strain) were exposed to different levels of Hcy(0?25?50?100 μmol/L?1?5?10 mmol/L)for 24 hours. And then cell viability was determined by MTT assay, and lactate dehydrogenase (LDH) released into the culture medium was measured to assess cell damage. The antigen levels of the soluble thrombomodulin shedded from cell surface molecules were measured in the culture supernatants by using ELISA (Diagnostica Stago, France). Furthermore, the total TM was measured by using cellular ELISA. Results:There were no signs of cell toxicity induced by Hcy (0-1mM) in terms of cell viability, LDH leakage and the shedding of sTM in culture media during 24 hours of incubation. The endothelial cells exposed to concentrations up to 5mM exhibited a decreased cell viability and higher level of Hcy (10mM) induced a significant cytotoxic effects: decreased cell viability, significant LDH leakage and increased sTM in culture media, compared with the control cells. The protein of TM was positive in all groups. Hcy promoted the production of TM in endothelial cells and the effect was significant when Hcy concentration was greater than 1mM. Conclusion:The low physical concentration of Hcy has no signs of cell toxicity but nonphysiological high homocysteine levels can result in a variety of cellular effects, which include the influence on the expression of TM.