【作者】 邓丽蕴； 黄香宜； 任吉存；

【Author】 Liyun Deng;Xiangyi Huang;Jicun Ren;School of Chemistry & Chemical Engineering, Shanghai Jiao Tong University;

【机构】 上海交通大学化学化工学院；

【摘要】 细胞内药物分子与靶标蛋白相互作用的研究对药物开发和疾病治疗有重要意义[1],但是目前还缺乏在活细胞内原位分析药物分子与靶标蛋白相互作用的有效方法。本文基于微流控芯片-荧光相关光谱(FCS)系统,建立了一种原位、实时检测单细胞内无标记药物分子与靶标蛋白相互作用的新方法。我们以筛选激酶c-Abl的抑制剂为模型[2],合成了Cy3标记的达沙替尼荧光探针(Cy3-dasatinb),将微流控芯片与FCS联用,实现了对细胞的在线培养、荧光标记、药物刺激和检测。我们系统研究了细胞内荧光探针与c-Abl结合以及药物作用下探针-靶蛋白复合物的解离,发现:(1)Cy3-dasatinb能与细胞内c-Abl激酶进行特异性结合(图1A)。(2)细胞内抑制达沙替尼与c-Abl激酶结合的不同药物的有效作用浓度存在显著差异(图1B)。(3)不同药物作用时,细胞内达沙替尼与c-Abl激酶的解离速率和解离程度各不相同(图1C)。同时我们建立了理论模型用于评价药物在活细胞内的抑制效果。更多还原

【Abstract】 A new method for in situ studying the interaction between drug-protein in single living cells was established based on the microfluidic chip-FCS system. Using Cy3-dasatinib as an affinity probe of kinase(c-Abl), a microfluidic chip combined with FCS was used for on-line cell culture, labeling, drug stimulation, and in situ detection. It was found that probe can specifically bind to intracellular c-Abl. Effective concentrations of different drugs to inhibit the binding of Dasatinib probe and c-Abl was significantly different in the cells. The dissociation rate probe-target complexes and the binding of probe-target were different for different drugs. The established model can be used to in situ evaluate the effect of drugs on the interaction of drug-target proteins within living cells.更多还原