基于核酸适体的细胞间通讯实时成像探针的研究

Tunneling nanotube (TNT) and TNT-like structures are cellular connections with fine and long membrane conduits. These TNT and TNT-like connections play important roles in cell-cell communication through directly transferring cellular cargos such as proteins and organelles between connected cells. Despite of significance of these connections, investigations of them lagged far behind due to the lack of biomarkers and specific probes; morphology and cytoskeletal components are currently the main criteria for classification and identification of these connections. Recently, the applicant has reported an aptamer named M17A2 which can specifically recognize a kind of TNT-like intercellular connections; and its target protein, selectively expressed on the intercellular connections, could be one of biomarkers of these TNT-like connections. Based on the above mentioned, we plan to optimize and modify the sequence and structure of aptamer M17A2; to purify and identify target protein using optimized aptamer; to investigate the molecular mechanism of the recognition between M17A2 and its target protein; to construct a novel aptamer probe for real-time dynamic imaging of TNT-like connections in living cells; and to study the mechanism of structure formation and biological functions of TNT-like connections by aptamer probe combined with molecular biological methods. Though the completion of these proposed work, novel methods to study the cell-cell communication through the TNT-like connections and its biological functions will be established; meanwhile, aptamer based techniques and methods for dynamic analysis of living cells will be developed.

隧道纳米管及其类似结构是一类细长精密管状的细胞间连接结构，可介导细胞间信息和物质的交流和传递，是细胞远程直接通讯重要途径。由于缺乏特征标志物和特异性探针，目前对该类结构的判断主要依赖形态学方法，因此对其形成的分子机制与生物学功能尚了解不多。申请人前期研究报道了核酸适体M17A2能特异识别隧道纳米管类似结构；其靶蛋白选择性表达于该结构上，是这类结构可能的蛋白标志物之一。基于此，本项目拟优化和改造M17A2，分离和鉴定M17A2的靶蛋白，研究分子识别机理，构建可用于活细胞隧道纳米管类似结构的实时动态成像分子探针；以构建的探针结合分子生物学手段，研究活细胞中该结构形成的分子机制，探索其在不同生长环境中的生物学功能。通过上述研究，在分子水平上建立活细胞间直接通讯动态过程和生物学功能的研究新方法；同时，发展核酸适体用于活细胞动态分析的技术和方法，拓展核酸适体在生物医学分析中应用范围。

本项目以核酸适体为主要研究工具，在分子水平上对活细胞通讯过程和相关生物学功能开展研究。我们利用已有的靶向肿瘤耐药细胞隧道纳米管类似结构的核酸适体M17A2和筛选获得的靶向神经细胞神经突的核酸适体yly21对细胞间通讯结构进行研究；首先，对核酸适体进行结构优化和改造，然后以这些核酸适体为亲和配基纯化靶蛋白，利用质谱技术进行蛋白质标志物的鉴定；同时，利用核酸适体yly21构建探针对神经突三维网状结构进行了成像分析。细胞通讯是肿瘤耐药性传递的重要途径，因此，我们对肿瘤耐药相关分子机制进行了研究，筛选了靶向耐紫杉醇卵巢癌细胞的核酸适体HF3-58并提取了其靶蛋白、开发了基于核酸适体的肿瘤耐药细胞复杂基质中的检测方法。此外，我们还对活细胞通讯过程相关物质如蛋白糖基化和microRNA等进行了分析与检测。上述研究成果不仅为后续发展基于核酸适体的活细胞动态成像新技术和新方法奠定了基础，也拓宽了核酸适体在生物医学分析中应用范围。