基于化学发光的DNA天然保护药物高通量筛选模型的研究

High performance and safe method to screening DNA protective natural products makes significance for DNA damage originated cancer, ageing and genetic disease . Here, we describe a novel strategy for monitoring of Fenton reagent or cigarette smoke-induced DNA oxidation damage and offer a method for screening natural products that protect DNA against reactive oxygen species(ROS) arising from environmental pollutants.The present protocol takes advantage of a fast and simple magnetic separation/mixing method and a highly sensitive chemiluminescence ELISA(CL ELISA). The DNA immobilized on the magnetic beads is attacked by the smoke，irradiation or metal ion in the absence or presence of natural products, and then damaged DNA is conveniently held by magnetic force, whereas the complex tobacco smoke matrix and any remaining compounds are completely eliminated by extensive washing, and possible interferences are thus removed and oxidative damage , 8-Hydroxy Deoxyguanosine (8-OHdG) and DNA single strand break(SSB), are then sensitively monitored by CL ELISA. A library of natural products will be screened and the effective compounds may be promising for the development of new drugs.In this project,we also need to investigate the mechanism, optimize the protocal and validate it by traditional cell and animal model. This innovative protocal based on magnetic separation/mixing method and CL ELISA is supposed to avoid the interference of the complicated impurity and lower the probability of "false positive". Moreover, the required sample is only trace and it can save more experimental cost. It will lead to a great potential to evaluate the genotoxicity of the environmental pollutant and to screen natural protective products for DNA with high-throughput.

高效地寻找DNA天然保护药物对预防DNA损伤引起的癌症、衰老、遗传疾病等具有重要意义。本课题以DNA单链断裂及8-羟基脱氧鸟苷（8-OHdG）为DNA损伤标志物，将微量DNA序列固定于具有特定功能的载体（磁性微球）上，并引入特异性高灵敏的酶联免疫化学发光(CL ELISA)检测手段，在分子水平上构建金属离子、辐射或吸烟等环境污染源对DNA 损伤评价及DNA天然保护药物筛选模型，研究模型中DNA损伤及天然药物作用的机理，优化模型并对筛选出的高效天然药物进行细胞、整体动物水平的验证，以确定其作用。模型创新性地偶合快速的载体分离技术与CL ELISA传感技术，将排除复杂成分对检测的干扰，为环境污染物遗传毒性的快速评价及预防药物的高通量筛选提供新思路。

高效地寻找DNA天然保护药物对预防DNA损伤引起的癌症、衰老、遗传疾病等具有重要意义。本课题以DNA单链断裂及8-羟基脱氧鸟苷（8-OHdG）为DNA损伤标志物，将微量DNA序列固定于具有特定功能的载体（磁性微球）上，并引入了特异性高灵敏的酶联免疫化学发光(CL ELISA)检测手段，在分子水平上构建了Fenton试剂介导的羟基自由基、吸烟等环境污染源对DNA 损伤评价及DNA天然保护药物的筛选模型，课题优化了模型并研究了模型中DNA损伤及天然产物作用的机理，应用模型筛选了上百种天然产物，对筛选出的高效天然产物进行了细胞、整体动物水平的验证，确定了其作用。模型创新性地偶合了快速的载体分离技术与CL ELISA传感技术，实验证明可以排除复杂成分对检测的干扰，为环境污染物遗传毒性的快速评价及预防药物的高通量筛选提供了新工具。