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Objective: dynamic observation of homocysteine ??(Homocysteine, Hcy) role of vascular endothelial cells after morphological and functional changes, as well as on endothelial cells actin G-actin and F-actin observed qualitative and quantitative analysis to explore Hcy actin through injury, and thus the possible mechanism of endothelial injury. Methods: Human umbilical vein endothelial cells (ECV-304) were cultured in vitro, the normal control group (Control), low-dose Hcy group (0.1mmol / L), medium dose Hcy group (0.5mmol / L), high-dose group Hcy (1.0mmol / L). Hcy were acting 6h, 12h, 24h, the inverted phase contrast microscope morphological changes of endothelial cells; using Rhodamine-Phalloidin labeled actin (F-actin), Alexa Flour488-Dnase Ⅰ labeled globular actin monomers (G -actin), laser scanning confocal microscope (laser scanning confocal microscope, LSCM) observed morphological changes of endothelial cytoskeleton and endothelial cells detected by LSCM skeleton G-actin, F-actin content, calculate the G / F ratio. Were used nitrate reductase, thiobarbituric acid assay conditioned medium of nitric oxide (NO), lipid peroxidation metabolites malondialdehyde (MDA) levels. Results: 1. Endothelial cell morphology: an inverted phase contrast microscopy revealed that the control group endothelial cells were spindle or round, showed a typical cobblestone appearance of cells arranged in compact. Hcy groups different doses endothelial cells were changed with the time and / or Hcy dosage, the cells showed shrinkage, cell gap increases, in the high dose group, even when the part of the cell necrosis. (2) endothelial cytoskeleton changes: (1) endothelial cell morphology skeleton: LSCM endothelial cells was observed in control group, F-actin band around the perimeter to form a dense cytoplasm some stress fibers. With increasing doses of Hcy, with the gradual emergence of peripheral dense fracture, dissipate, intracytoplasmic stress fibers shorter, thicker and eventually disappear. With time, endothelial cell F-actin rearrangement, peripheral dense band of local fracture occurred, while cytoplasmic stress fibers gradually increased thickening. Group G-actin endothelial cells distributed in the nucleus, with the time and / or increase the dose Hcy, G-actin and gradually spread to the membrane, the fluorescence intensity increases. (2) endothelial cytoskeleton G-actin content change: 6h, 24h, the different doses of Hcy group compared with the same period in the control group, G-actin content increased with the dose gradually increased; when at 12h, G-actin levels increased overall , but no significant dose-dependent manner. Normal control group and 0.1mmol / L Hcy group with time G-actin content increased, decreases; in Hcy 0.5mmol / L group and 1.0mmol / L group G-actin levels were gradually increased with time. (3) F-actin cytoskeleton of endothelial content change: 6h, 12h, the different doses of Hcy group compared with the same period in the control group, F-actin levels increased Hcy in the low-dose group, the highest content of F-actin; 24h time, F -actin content increased with the dose upward trend. High-dose Hcy group F-actin content gradually increased with time, other dose groups were first increased and then decreased, but the overall trend is increasing. (4) G / F ratios: 6h when different doses of Hcy group compared with the same period in the control group, G / F ratio increased, Hcy at a concentration of 0.5mmol / L, the highest ratio in the same period. 12h, 24h time, G / F ratios were increased as the dose increases. Each dose group with time G / F ratio increases. 3 endothelial cell function changes: (1) NO content change: different doses of Hcy group compared with the control group over the same period, as the dose increases, the NO content in each time period the overall declining trend, but Hcy at a concentration of 0.5mmol / L, a slight trend turning point. With time, NO content overall downward trend; at 12h, relative 6h, 24h speaking, NO high levels. (2) MDA content change: different doses of Hcy group compared with the control group over the same period, as the dose increases, the time period of MDA overall upward trend, with time, the content of MDA in each group also showed an upward trend. Conclusion: 1.Hcy not only damage endothelial cells and reduce endothelium-derived NO production, promote lipid peroxidation product MDA content increased, there are also significant impact on endothelial function. 2.Hcy endothelial cytoskeleton rearrangement and cytoskeletal proteins by changing the G-actin and F-actin content, and G / F ratio to participate in the regulation of endothelial cell morphology and function damage.
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