Moldovan N I, Radu A N, Simionescu N
Exp Cell Res. 1987 Jun;170(2):499-510. doi: 10.1016/0014-4827(87)90324-7.
A method using low concentrations of formaldehyde and dithiothreitol was applied to obtain 'right-side out' luminal plasmalemma-derived vesicles from bovine aortic endothelial cells (EC) in culture, and from human umbilical vein and bovine or porcine aortas perfused ex vivo with the vesiculation solution. Vesicle formation and shedding were examined by phase-contrast microscopy and by transmission (TEM) and scanning electron microscopy (SEM). Vesicles showed the characteristic trilaminar pattern of the unit membrane and did not contain cellular organelles. As detected in freeze-fracture preparations, vesicle membrane displayed intramembrane particles and filipin-detectable cholesterol. Like EC plasmalemma, vesicle surface was heavily stained by Ruthenium Red and bound under a normal pattern cationized ferritin and ferritin hydrazide. As indicated by lectin agglutination assays and by ultrastructural cytochemistry, vesicles maintained on their ectodomains glycoconjugates bearing monosaccharides such as N-acetyl-neuraminic acid, beta-N-acetylglucosamine and beta-D-galactose, and expressed 5'-nucleotidase activity. The electrophoretic profiles of externally disposed 125I-labelled polypeptides of vesicles were found to be similar to those of intact EC. Chemically-induced vesiculation appears as a suitable method to obtain EC plasmalemma for studying its composition and functions in various vascular beds.
一种使用低浓度甲醛和二硫苏糖醇的方法被应用于从培养的牛主动脉内皮细胞（EC）以及用囊泡化溶液进行离体灌注的人脐静脉和牛或猪主动脉中获取“外翻”的管腔质膜衍生囊泡。通过相差显微镜、透射电子显微镜（TEM）和扫描电子显微镜（SEM）检查囊泡的形成和脱落情况。囊泡呈现出单位膜特有的三层结构模式，且不含细胞器。在冷冻断裂标本中检测到，囊泡膜显示出膜内颗粒以及可被制霉菌素检测到的胆固醇。与EC质膜一样，囊泡表面被钌红强烈染色，并以正常模式结合阳离子化铁蛋白和铁蛋白酰肼。凝集素凝集试验和超微结构细胞化学表明，囊泡在外结构域上维持着带有N - 乙酰神经氨酸、β - N - 乙酰葡糖胺和β - D - 半乳糖等单糖的糖缀合物，并表现出5'-核苷酸酶活性。发现囊泡外部125碘标记多肽的电泳图谱与完整EC的电泳图谱相似。化学诱导囊泡化似乎是一种获取EC质膜以研究其在各种血管床中的组成和功能的合适方法。
