Yura Hirofumi, Ishihara Masayuki, Kanatani Yasuhiro, Takase Bonpei, Hattori Hidemi, Suzuki Shinya, Kawakami Mitsuyuki, Matsui Takemi
NeTech Inc. KSP West 300-H, Sakado 3-2-1, Takatsu, Kawasaki, Kanagawa 213-0012.
J Biochem. 2006 Apr;139(4):637-43. doi: 10.1093/jb/mvj077.
Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.
对用异硫氰酸荧光素（FITC）标记的合成半乳糖基聚合物、去唾液酸胎球蛋白和低密度脂蛋白（LDL）衍生物进行了流式细胞术分析，以确定各种类型细胞上的内吞受体表型，如去唾液酸糖蛋白（ASPG）和LDL受体。当将作为ASPG合成配体的FITC标记的半乳糖基聚苯乙烯（GalCPS）应用于大鼠肝细胞和人癌细胞（Hep G2和Chang Liver细胞）时，表面荧光强度根据细胞上的受体表达而变化。荧光强度源自FITC标记的GalCPS的钙依赖性结合。尽管用葡糖基聚苯乙烯（GluCPS）、胎球蛋白和LDL预处理不会改变荧光强度，但用（未标记的）GalCPS和去唾液酸胎球蛋白预处理会抑制荧光强度。流式细胞术使我们能够证明，添加17α-乙炔雌二醇后，FITC标记的LDL（由兔制备）的钙依赖性结合会增强LDL受体表达，而添加LDL受体单克隆抗体后该表达会受到抑制。基于FITC标记配体组合的结合效率表明，无需免疫活性抗体或放射性标记物质，即可用于对与内吞受体表达相对应的细胞类型和条件进行分类。此外，合成糖缀合物（GalCPS）被证明是一种基于细胞表型使用流式细胞术进行分类的灵敏且有用的标记物。
