利用AFM原位观察兔主动脉和主动脉瓣膜内皮细胞表面超微结构

目的利用原子力显微镜（AFM）原位观察正常新西兰白兔主动脉和主动脉瓣膜内皮细胞表面超微结构。方法取10只正常健康雄性新西兰白兔的新鲜主动脉和主动脉瓣膜组织，经磷酸盐缓冲液去除组织表面杂质后，置于1％的甲醛溶液中固定。在大体显微镜下，将固定好的主动脉和瓣膜组织粘附并展平在粘着有双面胶的载玻片上，并将主动脉的内膜面和主动脉瓣膜的动脉面置于上部，待样本干燥后，将其置于NanoSeope Ⅲa AFM载物台上，选择其中某个区域，在Tapping模式下进行扫描。结果AFM能够清晰的原位显示位于心脏主动脉瓣膜主动脉面和主动脉内膜上的内皮细胞表面的三维结构，主动脉内膜表面的内皮细胞大，分支多，排列稀疏，与血流方向平行；主动脉瓣膜表面的内皮细胞较小，分支少，排列紧密，与血流方向垂直。当扫描范围缩小时，能够观察到细胞表面颗粒样的微细结构。当扫描范围进一步缩小时，可见主动脉内皮细胞表面的颗粒样物呈沟回状，主动脉瓣膜内皮细胞表面的颗粒样物呈中央凹陷，周围隆起火山口样结构。结论AFM对主动脉和主动脉瓣膜的内皮细胞表面超微结构原位观察的方法是切实可行的。

In situ observation of surface structures of aorta and aorta valve endothelial cells with AFM

Objective To observe the surface structures of cardiovascular endothelial cells in situ with atom force microscope(AFM).Methods Fresh aorta and aortic valve were dissected from 10 healthy male New Zealand White rabbits after the animals were executed.Before fixed in 1% formaldehyde,the fresh tissues were washed 3 times in the buffer phosphate solution.Under general microscope,the fixed aorta or valve was spread on the double side stick tape which had already stuck on the glass slide.The intima of aorta or the aorta side of valve was towards upside.Then the specimen was dried under 37 degrees centigrade in attemperator and was washed again with pure water.After dried again,the specimen was loaded on the platform of NanoScope IIIa AFM and scanned in tapping mode with the scanning speed of 0.5 HZ.Results The surface structures of endothelial cell could be observed clearly in situ with AFM.Endothelial cells on aorta were large and branched which arranged sparsely and parallel to the direction of blood flow,whereas endothelial cells on aorta valve surface were small and less branched which arranged intensively and vertical to the direction of blood flow.When the scanning area was dwindled,granular ultra-structure could be observed on the surface of endothelial cells.When the scanning area was dwindled further,fissure and convolution could be seen on the surface of granules from aortic endothelial cells. Centre cavity and surrounding swelling volcano-like structure could be seen on the surface of granules from endothelial cells of aortic valves.Conclusion It's feasible to observe the surface ultra-structures of cardiovascular endothelial cells in situ with AFM.