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目的:通过动态观察同型半胱氨酸(Homocysteine,Hcy)作用后的血管内皮细胞功能的变化,以及对内皮细胞骨架肌动蛋白G-actin和F-actin的定量分析,探讨骨架肌动蛋白在Hcy损伤内皮中的作用及机制。方法:将体外培养的人脐静脉内皮细胞株(ECV-304)分为正常对照组、低剂量Hcy组(0.1mmol/L)、中剂量Hcy组(0.5mmol/L)和高剂量Hcy组(1.0mmol/L),观察各组细胞培养24h时其细胞骨架和相关功能的变化;中剂量Hcy组及高剂量Hcy组还观察了6h、12h时的相应变化。分别采用硝酸还原酶法、硫代巴比妥酸法检测各组条件培养基中一氧化氮(NO)、脂质过氧化代谢产物丙二醛(MDA)的含量。用Rhodamine-Phalloidin标记纤维状肌动蛋白(F-actin),Alexa Flour488-DnaseⅠ标记球状肌动蛋白单体(G-actin),激光扫描共聚焦显微镜(LSCM)检测内皮细胞骨架G-actin、F-actin含量,并计算G/F比值。结果:随着Hcy作用时间的延长或剂量的增大,NO含量总体呈下降趋势,而MDA含量增高;G-actin、F-actin总体含量及G/F比值上升,并呈一定的时间、剂量相关性。结论:Hcy可通过改变骨架蛋白G-actin和F-actin含量,以及G/F比值引致内皮细胞功能的损伤,这也可能是其诱导动脉粥样硬化早期变化的重要机制之一。
OBJECTIVE: To observe the changes of endothelial cell function after the observation of Homocysteine (Hcy) and the quantification of actin G-actin and F-actin in endothelial cells, Hcy damage the role of the endothelium and mechanism. Methods: Human umbilical vein endothelial cell line (ECV-304) cultured in vitro was divided into normal control group, low dose Hcy group (0.1mmol / L), middle dose Hcy group (0.5mmol / L) and high dose Hcy group 1.0 mmol / L). The changes of cytoskeleton and related functions in each group were observed at 24 h. The corresponding changes of Hcy group and Hcy group at 6h and 12h were also observed. Nitric acid reductase and thiobarbituric acid were used to detect the content of nitric oxide (NO) and malondialdehyde (MDA) in the conditioned medium of each group. F-actin, Alexa Flour488-Dnase I labeled G-actin and LSCM were used to detect the expression of G-actin, F -actin content, and calculate the G / F ratio. Results: With the prolongation of Hcy action time or dose, the content of NO decreased and the content of MDA increased. The content of G-actin, F-actin and the ratio of G / F increased, and the time and dosage Correlation. CONCLUSION: Hcy can induce endothelial dysfunction by changing the content of G-actin and F-actin, as well as G / F ratio, which may be one of the important mechanisms of Hcy in inducing the early changes of atherosclerosis.