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以辣根过氧化物酶标记人LDL、兔抗人LDLIgG或羊抗兔IgG抗体作为示踪剂,作用于培养的人胚肺细胞,经DAB-H_2O_2显色,超薄切片,电镜观察,结果表明: (1)细胞能与很低浓度LDL-HRP产生特异性结合,显示LDL受体的高亲和力。 (2)兔抗人LDLIgG-HRP与已结合在受体上的LDL的结合可被未标记的兔抗人LDLIgG封闭,而封闭后以羊抗兔IgG-HRP作用又显示特异性反应,这种反应只能在已有LDL结合的细胞膜上显示,IgG不能阻断这种反应。 (3)肝素或高浓度LDL对LDL-HRP与受体的结合产生竞争性抑制;事先经高脂培养,这种结合也受到抑制(反馈性抑制)。 (4)细胞对游离HRP不显示特异性高亲和力的结合。 (5)生长在不含LDL-HRP是培养液中的细胞也无这种特殊性反应。上述纳果说明,LDL-HRP是结合于质膜表面有外被区特异性的、高亲和力的LDL受体上。
Human LDL, rabbit anti-human LDL IgG or goat anti-rabbit IgG were labeled with horseradish peroxidase (HRP) as tracer and cultured in human embryonic lung cells. The cells were stained with DAB-H 2 O 2, ultrathin sections and electron microscopy. The results showed that: (1) The cells could specifically bind LDL-HRP at very low concentration, showing the high affinity of LDL receptor. (2) The binding of rabbit anti-human LDLIgG-HRP to LDL bound to the receptor can be blocked by unlabeled rabbit anti-human LDLIgG, which shows a specific reaction with goat anti-rabbit IgG-HRP after blocking The reaction can only be shown on the membranes of existing LDL-bound cells, which can not block this reaction. (3) Heparin or high concentration of LDL competitively inhibits the binding of LDL-HRP to the receptor; this binding is also inhibited (feedback inhibition) by prior high-fat culture. (4) Cells do not show specific, high-affinity binding to free HRP. (5) Cells grown in culture medium without LDL-HRP also do not have this particular response. The above results suggest that LDL-HRP binds to the outer membrane-specific, high-affinity LDL receptor on the plasma membrane surface.