基于信号放大的肺癌、肝癌细胞内多种活性分子同时高灵敏荧光成像新方法研究

Being the most common tumor forms, lung cancer and liver cancer cause very high mortality rate. To further research the signal pathway in the occurrence and development of lung cancer and liver cancer and the process of drug treatment, improve the accuracy of early diagnosis, it is necessary to simultaneously monitor the concentration and the temporal and spatial variation of multiple intracellular molecules. But, limited to the low concentration of some molecules in living cells, it is difficult to directly detect them. Thus, it is of great significance to amplify the signal of the molecules at low level and make highly sensitive fluorescent imaging of lung cancer and liver cancer cells. Here, we present a novel method for simultaneous fluorescent imaging of multiple biological molecules in living lung cancer and liver cancer cells based on signal amplification. In the proposed method, functional DNAs will be used for the specific recognization of the target molecules including proteins, miRNA and metal ions; signal amplification will be realized based on kinds of DNA reactions, such as RCA and HCR; nanomaterials and biomaterials will be chosen as cellular carriers for probe transfer. The method can simultaneously monitor the cellular multiple biological molecules on site. Using this method, we will also explore the relationship of the cellular intake of Zn2+ and Cu2+ in lung cancer and liver cancer cells as well as the changes of the metal ions and related other molecules in the lung cancer cells after an induction of drug. The proposed method will provide a powerful tool for the researches of signal pathway during the occurrence and development of lung cancer and liver cancer, also provide new pathway for the accurate early diagnosis of these diseases.

肺癌和肝癌是致死率极高的泛发肿瘤。为了研究肺癌和肝癌发生、发展和药物作用过程中多分子参与的信号通路，提高此类疾病早期诊断的准确性，在活细胞水平上同时监控肺癌和肝癌细胞内多种分子的浓度和时空变化十分必要。由于细胞内生物分子浓度差异大，低浓度分子的信号难以直接获取，因此发展基于信号放大的肺癌、肝癌细胞内低浓度分子同时高灵敏成像方法具有重要的意义。本项目拟利用功能DNA进行细胞内多种分子的特异性识别；构建多种DNA扩增反应进行目标分子荧光信号放大；设计新型的纳米材料/DNA组装体进行细胞内探针输送，实现活细胞内多种生物分子的高灵敏荧光成像。基于该方法，我们将研究肺癌和肝癌细胞内不同金属离子摄取的相互关系及药物调控肺癌细胞内金属离子变化引发下游生物分子的变化。该方法将为我们进一步研究肺癌和肝癌发生、发展和药物治疗相关的信号通路提供有力的工具，为肺癌和肝癌的有效早期诊断提供新的途径和依据。

项目申请者聚焦于细胞内具有重要生理、病理意义的活性分子，针对细胞内多种生物分子的同时高灵敏成像和定量检测等关键科学问题，开展了系列新方法、新技术和新装置研究，为识别细胞内重要的生物分子，研究多种生物分子的含量、功能等提供了重要的测量方法与工具。主要研究内容如下：构建了用于单细胞操控和分析的微流控芯片系统，发展了多种高通量、高精度单细胞操控技术；在此基础上，发展了肿瘤细胞膜蛋白、miRNA、金属离子等多种生物分子专一识别、荧光信号放大、单细胞水平多种分子同时成像和定量分析新方法；并将所发展的肿瘤细胞内多种活性分子同时高灵敏检测方法应用于临床检测研究中。在项目资助下，项目负责人在JCR杂志发表高水平学术论文16篇，申请国家发明专利5项。在该项目研究基础上，项目负责人获批国家自然科学基金重大研究计划培育项目一项（91859111），山东省自然科学杰出青年基金一项（ZR2019JQ06），获评山东省泰山学者青年专家。

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