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目的:研究Antiflammin-1(AF-1)与其受体在活细胞表面的结合特性。方法:激光共聚焦显微镜观察标记的AF-1与NIH 3T3细胞的结合;基于流式细胞分析的配基结合方法对不同方法标记的AF-1与细胞表面的受体结合动力学参数进行分析。结果:激光共聚焦显微镜观察提供了AF-1与活细胞结合的证据;流式细胞分析结果表明,过量未标记的AF-1可以竞争标记的AF-1与细胞表面的结合。Scatchard作图为线性,符合简单位点系统特征,Kd值在29.19~46.33nM之间,最大结合容量Bmax为93.29 4.267 RFI。结论:AF-1与NIH 3T3在活细胞表面呈高亲和力结合。本研究结果有助于揭示AF-1与其受体在生理条件下的结合特性。
Objective: To study the binding characteristics of Antiflammin-1 (AF-1) and its receptor on the surface of living cells. Methods: Confocal laser scanning microscopy was used to observe the binding of labeled AF-1 to NIH 3T3 cells. The ligand binding kinetic parameters of AF-1 labeled with different methods and cell surface were analyzed by flow cytometry. Results: Confocal laser scanning microscopy provided evidence of binding of AF-1 to living cells; flow cytometric analysis showed that excess unlabeled AF-1 competed for binding of labeled AF-1 to the cell surface. Scatchard plots are linear and consistent with simple locus system characteristics with Kd values between 29.19 and 46.33 nM and maximum binding capacity Bmax of 93.29 4.267 RFI. Conclusion: AF-1 and NIH 3T3 bind with high affinity on the surface of living cells. The results of this study will help to reveal the binding properties of AF-1 and its receptors under physiological conditions.