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目的:研究抗体偶联药物T-DM1与乳腺癌细胞SKBR3表面人表皮生长因子受体2(HER2)的相互作用及内吞机制。方法:采用基于表面等离子共振(SPR)原理的Biacore方法测试配体T-DM1与受体HER2的相互作用,通过流式细胞仪测定药物在体外内吞清除率,利用激光扫描共聚焦显微镜原位检测药物在细胞中的内吞过程。结果:T-DM1与HER2具有高亲和力,ka为6.234×10~6mol/(L·s),kd为3.077×10~(-4)/s,KD为4.936×10~(-11)mol/L,流式细胞实验证明受体与配体的结合具有饱和性;利用免疫荧光方法检测到T-DM1在SKBR3细胞内的消除呈时间-剂量依赖关系,实验表明4 h内荧光强度减低32%,48 h时降低约90%;共聚焦显微镜实验表明T-DM1先与SKBR3细胞表面的HER2受体结合,进入细胞内,反应1 h到达溶酶体,48 h时在显微镜下已消失。结论:T-DM1与HER2具有高亲和力及结合饱和性,在1 h内通过HER2介导内吞进入细胞,随后定位于溶酶体,最后于48 h裂解。
AIM: To investigate the interaction and endocytosis mechanism of T-DM1, an antibody-coupled drug, and human epidermal growth factor receptor 2 (HER2) on the surface of breast cancer SKBR3 cells. Methods: The interaction between ligand T-DM1 and receptor HER2 was measured by the Biacore method based on the principle of surface plasmon resonance (SPR). The clearance rate of endocytosis in vitro was determined by flow cytometry. In situ scanning laser scanning confocal microscopy Detection of drug endocytosis in the cell process. Results: T-DM1 had high affinity with HER2, with ka of 6.234 × 10-6mol / (L · s), kd of 3.077 × 10-4 / s and KD of 4.936 × 10-11 mol / L, flow cytometry showed that the binding of the receptor with the ligand saturation; detected by immunofluorescence T-DM1 elimination in SKBR3 cells in a time-dose-dependent manner, the experiment showed that fluorescence intensity within 4 h reduced by 32% , And decreased by about 90% at 48 h. Confocal microscopy showed that T-DM1 bound to HER2 receptor on the surface of SKBR3 cells first and then reached the lysosomes within 1 h and disappeared under the microscope 48 h later. Conclusion: T-DM1 and HER2 have high affinity and saturation of binding, and enter into cells by HER2-mediated endocytosis within 1 h, then locate in lysosome and finally cleaved at 48 h.