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PHARMCOLOGICAL STUDY ON THE 3-DIMENSIONAL ULTRASTRUCTURE OF A BLOOD PLATELET

血小板三维超微结构的药理学研究

基本信息

批准号：
63571098
负责人：
NISHIO Hiroaki
金额：
$ 1.28万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

In order to examine the 3-dimensional ultrastructure of a blood platelet with a high voltage electron microscope (HVEM), we had been mainly prepared the sample by critical point drying method, which give us somewhat unsatisfactory results as to its reproducibility. In this study, we established the method using freeze drying method, which did not include the medium exchange steps with organic solvent and liquid carbon dioxide. Thus, platelets, prefixed with glutaraldehyde, were attached to polyvinylformal sheet-mesh which had been coated with poly-L- lysine. They were post-fixed with glutaraldehyde and osmium tetroxide, and were stained with uranyl acetate. After rapid freezing with liquid nitrogen, they were dried in a tissue freeze dryer (EMSCOPE FD-500), and observation was made with a HVEM (HITACHI, H-1250 M), results of which were compared with that had been obtained by the ultra-thin sectioning method using a conventional electron microscope. Dense bodies, serotonin storage sites, was clearly observed as well as circumferential bands of microtubules. We could also identify alpha-granules and mitochondria, the characteristic appearance of which was somewhat obscure. Glycogen granules, however, were not identified. Furthermore, intracellular canalicular system and open canalicular system were 3-dimensionally analyzed by the stereo- paired images. We also analyzed the effect of various pharmacological modification, including serotonin, cold stress and some cell surface tracers such as ruthenium red. The work with a HVEM was done under the joint study program of National Institute for Physiological Sciences (Okazaki, Japan).

为了用高压电子显微镜（HVEM）检查血小板的三维超微结构，我们主要采用临界点干燥法制备样品，但其重现性有些不太令人满意。在本研究中，我们建立了使用冷冻干燥法的方法，该方法不包括有机溶剂和液态二氧化碳的介质交换步骤。因此，以戊二醛为前缀的血小板被附着到已涂有聚-L-赖氨酸的聚乙烯醇缩甲醛片网上。它们用戊二醛和四氧化锇进行后固定，并用乙酸双氧铀染色。用液氮快速冷冻后，在组织冷冻干燥机（EMSCOPE FD-500）中干燥，并用HVEM（HITACHI，H-1250 M）进行观察，将其结果与使用传统电子显微镜的超薄切片方法获得的结果进行比较。可以清楚地观察到致密体、血清素储存位点以及微管的圆周带。我们还可以识别α颗粒和线粒体，其特征外观有些模糊。然而，没有鉴定出糖原颗粒。此外，通过立体配对图像对细胞内小管系统和开放小管系统进行3维分析。我们还分析了各种药理修饰的效果，包括血清素、冷应激和一些细胞表面示踪剂（例如钌红）。 HVEM 的工作是在国家生理科学研究所（日本冈崎）的联合研究计划下完成的。