原子力显微镜观测人血小板与Ⅰ型胶原膜相互作用过程中的形态学变化  

Morphological changes of human platelets after interaction with type Ⅰ collagen films under atomic force microscope

作　　者：胡怡[1] 蔡继业[1] 吴扬哲[1] 王小平[2] 

机构地区：[1]暨南大学化学系,广东省广州市510632 [2]暨南大学第一附属医院麻醉科,广东省广州市510632

出　　处：《中国组织工程研究与临床康复》2007年第9期1647-1650,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30230350;30540420311)~~

摘　　要：目的:利用原子力显微镜从可视化的角度观察人血小板与Ⅰ型胶原膜相互作用后受激活化过程中的形态学变化。方法:实验于2006-06/08在暨南大学生物纳米技术实验室完成。①人血小板的提取:新鲜血液由健康无服药志愿者提供。抗凝剂(38g/L枸橼酸钠)与血液以1∶9的比例800r/min离心5min,将上层血浆以3000r/min离心10min,弃上清,加入生理盐水以3000r/min离心洗涤3次留沉淀。37℃下静置30min。所有操作均在室温下进行,pH为7.4。②Ⅰ型胶原膜的制备:把新鲜剥离的带负电的云母浸入NiCl2溶液中,使其表面带正电荷,通过静电作用将Ⅰ型胶原组装其上,室温下干燥。③血小板形态的观测:将新分离的血小板均匀铺展在膜材上与之作用,作用后用25g/L戊二醛固定10min,再用超纯水漂洗,室温下干燥后原子力显微镜观测。结果:①Ⅰ型胶原膜的表面形貌观察结果:胶原膜呈网孔状,胶原纤维清晰可辨;未受激血小板表面形貌观察结果:为了排除血小板在分离等前处理过程中受到激活的可能,对直接用戊二醛固定的血小板进行成像发现:约95%的血小板未被激活,血小板呈分散分布。对单个血小板的扫描成像发现血小板呈扁平圆盘状,表面比较平滑,血小板之间较分散,无聚集及伪足等活化标志。②Ⅰ型胶原膜上血小板形貌观测结果:与I型胶原作用后立刻用原子力显微镜观测,发现血小板聚集现象并不显著,仅在小范围内出现了交联聚集的现象。通过扫描样品上不同位置的血小板聚集体,发现血小板表面有许多呈平行排列的纤维束,它们把血小板整齐的连接起来,且血小板间层层相叠;这些嵌入在纤维蛋白原网内的血小板,大多保持了圆盘状的结构,但表面都分布着颗粒状或纤维条状物质;与Ⅰ型胶原作用1h后用原子力显微镜观测,发现单个血小板大多处于活化过程中的树突型,伸出了伪足,并有着彼此�AIM： To simulate the platelet＇s activation process of human, and to visualize the morphological changes of platelet after interacted with type I collagen film with atomic force microscope （AFM）. METHODS： The experiment was accomplished in the Bio-nanotechnology Laboratory of Jinan University between June and August 2006. （1）harvesting the platelets from the whole blood （contributed by healthy donor）： 38 g/L of Sodium Citrate （anticoagulant） and whole blood were mixed at the volume ratio of 1：9, and the mixture centrifuged at 800 r/min for 5 minutes, and extracted the platelet rich plasma （PRP）. Then centrifuged the PRP at 3 000 r/min for 10 minutes, finally washed the precipitate three times with saline, and incubated the precipitate for 30 minutes at 37 %. All of the processes were operated at room temperature and pH of 7.4. （2）preparation of type I collagen film： The newly cleaved mica was immersed into the NiCl2 solution to make the mica surface positively charged, and assembled the type Ⅰ collagen film on it through electrostatic interaction and air-dried; （3）observation of the platelets： The newly prepared platelets were spread on the type Ⅰ collagen film, and used 25 g/L of glutaraldehyde to fix the platelets for 10 minutes, then the ultrapure water was used to rinse the sample and air-dried, finally AFM was used to scan the sample. RESULTS： （1）The morphology of the type Ⅰ collagen film： Collagen film presented mesh-like structure, and the collagen fibers could be distinguished cleady. The surface morphology of the un-stimulated platelets： To avoid the possibility of platelets stimulated by the pretreatments, the platelets only treated with glutaraldehyde after extraction were imaged and found that about 95% platelets stayed quiescent stage and distributed scatteredly. The platelets kept fiat disk-like in shape, and the surface was relatively smooth. Aggregates and pseudopodia that could indicate the activation of platelets could not be seen on th

关 键 词：Ⅰ型胶原 血小板 纤维蛋白原 形态学结构 原子力显微镜 

分 类 号：R318.1[医药卫生—生物医学工程]