高脂饮食及阿托伐他汀干预对主动脉瓣膜内皮细胞表面超微结构的影响

Effects of high cholesterol diet and parallel atorvastatin treatment on the surface structures of aortic valve endothelial cells

目的利用原子力显微镜观察高脂饮食及阿托伐他汀干预对主动脉瓣膜内皮细胞表面形态结构的影响。方法新西兰纯种雄性白兔60只,随机分为对照组、高脂饮食组、高脂饮食+阿托伐他汀干预组,每组各20只。分别于实验开始及第2、4、6、8周末5个时间点,将上述3组动物各随机处死4只,取其心脏主动脉瓣膜,标本经处理后,置于原子力显微镜下扫描观察。结果随着高脂饮食时间的延长,高脂饮食组瓣膜表面内皮细胞的排列从规则的栅栏状密集的排列,逐渐过渡到无序紊乱的疏松排列。细胞形态从长梭形逐渐过渡为短圆形,细胞之间的间隙逐渐扩大。当AFM在内皮细胞表面的扫描范围进一步缩小时,随着高脂饮食时间的延长,内皮细胞表面大小一致,均匀排列的球形隆起结构逐渐变得低平、融合、减少;高脂饮食及阿托伐他汀干预组内皮细胞的改变介于对照组和高脂饮食组两组之间。结论利用AFM能够在瓣膜组织原位清晰显示损伤保护作用后,内皮细胞膜表面三维超微结构的变化;高脂饮食可以使主动脉瓣膜内皮细胞的排列、分布、形态发生明显改变,为后期与高脂血症有关的瓣膜疾病,例如钙化性主动脉瓣膜狭窄的发生提供条件。阿托伐他汀能够缓解高脂饮食所致的主动脉瓣膜内皮细胞形态结构的改变。

Purpose To observe the effects of high cholesterol diet and parallel atorvastatin treatment on the surface structures of aortic valve endothelial cells with atomic force microscope （AFM）. Methods New Zealand white rabbits （ n = 60） were randomly divided into 3 groups： control group （n =20）, fed with normal diet; high cholesterol diet group （n =20）, fed with 1% （wt/wt） cholesterol diet; and high cholesterol diet plus atorvastatin group（ n = 20）, fed with 1% （wt/wt） cholesterol diet plus atorvastatin （4 mg/kg per day）. After feeding for 0,2, 4, 6, or 8 weeks, 4 rabbits selected randomly from each groups were executed separately. Fresh aortic valve was dissected from the hearts. Before fixed in 1% formaldehyde buffer phosphate solution, the fresh valve was washed 3 times in the buffer phosphate solution. Under light microscope, the fixed valve was spread on the double side stick tape which had already been stuck on the glass slide. The intima of the aorta side of valve was towards upside. Then the specimen was dried under 37 degrees centigrade in attemperator and was washed with pure water. After dried again, the specimen was loaded on the platform of NanoScope In a AFM and scanned in tapping mode with the scanning speed of 1 - 2 Hz. Results With prolonged period of feeding high cholesterol diet time, arrangement of endothelial cells changed from regular palisade-like intensive alignment to disorder scattered alignment. The shape of endothelial cell transformed from long fusiform to short round shape. Interspaces between endothelial cells were enlarged. When the scanning area of AFM on the surface of endothelial cells was decreased further, uniform arranged globular bulged structures on the surface of endothelial cells became low, fused, and diminished gradually. The extent of structure changes of endothelial cells in high cholesterol diet plus atorvastatin group were between those of the other two groups. Conclusions AFM, with the merit of high resolution and easy sample preparation, could finely display the 3-dimentinal morphology of the surface of aortic valve endothelial cells. High cholesterol diet could influence the arrangement, distribution, and shape of endothelial cells, which might be the basis for later valve diseases, as calcific aortic valve stenosis, and treatment with atorvastatin could modifies this transformation.