稳态反应氧族介导肿瘤多药耐药的量效关系及机制研究

This project focuses on the difference between multidrug resistant cell and sensitive cell to find that the formation and maintenance mechanism of multidrug resistance may due to the reactive oxygen species (ROS). Firstly, the multidrug resistant cell lines and clinical samples with their parent sensitive ones are utilised to validate the common difference of ROS level. Secondly, pairs of cell models which have different levels of steady ROS will be built by long term incubation with oxidant or antioxidant. The high content living cell imaging system (HCS) could be used as a real-time monitor. Therefore, the specific dose-effect relationship between ROS content and multidrug resistance will be discovered, for providing the regulation direction of ROS content to reverse multidrug resistance. Thirdly, western blot, Elisa and the HCS will be applied to screen the multidrug resistant specific signal transduction molecules which are sensitive to ROS. Then, the inhibitors and siRNA technicals will help to clarify how ROS regulates the function and expression of P-gp as well as the and the synthesis of GSH. It will elucidates the mechanism of ROS mediated multidrug resistance. These findings will further reveal the mechanism of multidrug resistance, and facilitate to find new targets for multidrug resistant reversal, thereby to provide experimental and theoretical reference for a new strategy of drug therapy with more safety and efficiency.

本项目关注肿瘤多药耐药细胞和敏感细胞的差异，发现反应氧族（ROS）可能是耐药形成和维持的重要机制。首先选用成对的细胞株与临床样本作为研究对象，验证ROS含量差异的共性；然后以长期培养方式建立有不同稳态强度的ROS的成对细胞模型，辅以高内涵活细胞成像系统进行动态监控，从而揭示ROS含量与多药耐药之间较为精准的量效关系，为逆转多药耐药提供ROS的调控范围；之后利用western blot法、Elisa法和高内涵活细胞成像法等实验手段，有针对地筛选出ROS敏感的、具有多药耐药特异性的信号转导分子，在此基础上，通过抑制剂、基因沉默技术的靶向定位，理清ROS调控谷胱甘肽合成和P-糖蛋白功能、表达的信号转导过程，阐明ROS介导多药耐药的机制。研究结果有助于进一步揭示多药耐药的机理，并为耐药肿瘤的药物治疗寻找新的靶点、为形成更安全有效的治疗策略提供实验参考和理论依据。

肿瘤多药耐药表型繁多、机制复杂，信号转导通路呈联网性紊乱，迄今为止，相关基础研究尚未能阐明其核心靶点，临床治疗也因此裹足不前。本项目基于耐药肿瘤细胞与敏感肿瘤细胞间细胞内反应氧族的含量差异立题，通过双氧水/谷胱甘肽单因素造模20周以上，证实了双向稳态性调控环境反应氧族可诱导以MCF-7为代表的实体瘤细胞产生多药耐药，而对K562为代表的白血病细胞作用相对较弱。进一步机制研究以MCF-7细胞为研究对象，通过关注耐药细胞和敏感细胞间差异表达的功能分子，分别揭示了双向调控反应氧族诱导多药耐药产生机制的异同。靶点主要涉及：以P-gp、MRP1为代表的耐药蛋白；以GSTπ、Catalase、SOD、GPx、GSH为代表的抗氧化系统；以c-Myc、NF-κB为代表的增殖/凋亡控制分子等。研究成果主要体现为：通过调控反应氧族成功探索出一种新的肿瘤多药耐药细胞株建立方法，并以MCF-7/ROS为代表初步阐明了其耐药机制、绘制了相关经典耐药表型间的信号网络。本研究或为揭示多药耐药的核心机制、寻找具有潜在临床意义的药物靶点提供支持；实验结果同时也可为与P-糖蛋白、氧化应激相关的其他疾病的机制研究提供参考。