基于单粒子精确照射和SELEX技术筛选辐射损伤指示物适配体研究

Individial Radiation damage Indicator is very important for rapid diagnosis and treatment in nuclear safety accidents or nuclear terror attacks. The distribution of Particles in radiation samples by traditional radition methods is non uniform, so it is difficult to measure the damage of radiation. In this project, we will development a screening methods based on accurate radiation technology, FACS technology and cell index enrichment of the ligand system evolution technology (Cell-SELEX). Previously we had been built the single-particle microbeam device - CAS-LIBB. In order to acquire the radiation damaged cells for Cell-SELEX, we will investigate the relationship of precise dose radiation and radiation damage biological endpoint such as double strand break and micronucleus. Then these damaged cell (AG1522, a human skin fibroblast cells) will be sorted by BD FACS sorter as targets in SELEX and aptamers will be acquired by Cell-SELEX screening processes. Aptamers are molecular modified by fluorescence and streptavidin in order to indicate which cells were irradiated and separate the biomolecule from radiation damage cells in future. We also will build an accurate radiation damage cells diagnosis model based on application of these indicators. All of these work will provide important experimental data for the rapid dection in nulear safety accident and clinical application and explore the new methods to seek for radiation biomarkers.

辐射损伤指示物对于快速诊断在核安全事故或核恐怖袭击中受到辐射损伤程度和个体治疗至关重要。传统的照射方式由于粒子分布的非均匀性，难以确定针对特定剂量的辐射损伤。本项目申请基于我们已建成的单粒子微束辐照技术，通过精确定点定量照射人的皮肤成纤维细胞AG1522，建立特定辐射损伤生物学终点与照射剂量的关系；在此基础上，利用细胞指数富集的配体系统进化技术（Cell-SELEX），结合流式细胞分选技术，通过人工定向进化筛选出辐射损伤细胞的适配体（Aptamers），进一步通过分子修饰，开发辐射损伤的适配体指示物（Indicator），建立精确辐射细胞损伤快速诊断模型。探索辐射损伤快速检测和辐射生物标志物开发的新途径，为核安全事故中的快速检测及临床应用和辐射生物学学研究提供重要的实验数据。

在辐射生物学效应研究中，利用生物指标分析的方法来评估受照射剂量已经成为辐射生物学和辐射防护学研究中最受关注的领域。我们利用精确定位照射细胞照射技术对SELEX技术筛选生物辐射标记物的DNA适配体方法进行深入研究。首先研究了粒子精确辐照剂量与细胞凋亡、线粒体发生、TFAM表达等生物学终点及时间关系，针对各种辐射剂量和孵育时间与辐射早期生物学效应，确定正负筛选方法，通过筛选过程优化，获得一系列辐射指示DNA适配体。另一方面，针对生物辐射标记物低丰度的问题，通过精确照射方法研究大量依赖辐射剂量的生物分子，设立单一目标靶分子Human-IL6和HMGB1进行筛选，通过流式分选方法将两种蛋白成功在H1299细胞进行细胞表面展示，针对该细胞高表达的IL6和HMGB1进行Cell-SELEX筛选，设计并优化筛选过程，获得有效的DNA适配体序列。