甲基乙二醛对人血管内皮细胞的毒性作用

Cytotoxic effect of methylglyoxal on cultured human vascular endothelial cells

目的 探讨甲基乙二醛(methylglyoxal,MGO)对人血管内皮细胞的毒性作用及其机制。方法 体外培养的人脐静脉血管内皮细胞与不同浓度MGO共同孵育后,通过形态学观察和原位末端标记(TUNEL)法检测细胞凋亡,并以荧光Annexin-V及碘化丙碇(PI)染色,经流式细胞仪定量检测凋亡细胞和死亡细胞。细胞内氧化水平以氧化敏感的荧光染料2,7-二氢二氯荧光素(DCFH)染色,用流式细胞仪测定。结果 MGO刺激后,内皮细胞形态和超微结构出现凋亡的特异性变化,并且TUNEL染色阳性,流式细胞仪测定显示凋亡细胞和死亡细胞数量与MGO呈时间和剂量依赖关系;同时,MGO刺激后细胞内氧化水平明显升高,其时效与量效关系与细胞凋亡和死亡的变化相似。抗氧化剂(N-乙酰半胱氨酸和丙丁酚)及羰基化合物清除剂(氨基胍)对MGO引起的细胞氧化应激和细胞毒性具有抑制作用。结论 MGO对人血管内皮细胞具有直接的毒性作用,其机制可能是诱导细胞氧化应激。这些结果提示,活性羰基化合物蓄积可能与慢性肾功能衰竭和糖尿病血管并发症的形成有关。

Objective To elucidate the effect of methylglyoxal (MGO) on human vascular endothelial cells (VECs). Methods VECs were isolated from human umbilical vein and cultured with MGO in vitro. Apoptosis was confirmed by morphologic changes and TUNEL assay, and quantitated by flow cytometer using FITC Annexin-V and propidium iodid (PI) staining. Intracellular oxidative levels were measured by flow cytometric assay using an oxidant sensitive dye2, 7-dichlorefluoresin (DCFH). Results Apoptosis occured when VECs were incubated with MGO, evidenced by morphological changes and increased proportion of TUNEL positive cells. Apoptosis and death induced by MGO were both in a time- and dose-dependent manner. The level of intracellular oxidation increased simultaneously. The oxidation and cytotoxic effect of MGO on VECs were partly inhibited by carbonyl scavanger (aminoguanidine) and oxidative inhibitor( N-acetylcysteine and probucol) . Conclusion MGO induces apoptosis of VECs by increaseing intracellular oxidative level, which can be partially blocked by either carbonyl scavanger or antioxidants. These results indicate that accumulation of reactive carbonyl compounds may be involved in the pathogeneses of vascular diseases seen in chronic renal failure and diabetes.