基于无酶信号放大的AD相关miRNAs活细胞内原位分析

Baise is an important aluminum industrial base in Asia, high aluminum exposure is closely associated with Alzheimer's disease (AD). miRNAs are potential biomarkers and therapeutic targets, they play pivotal roles in a diverse range of AD processes. Thus, it is crucial to develop highly sensitive in situ analysis of AD related miRNAs in living cells. However, in situ analysis of miRNAs in living cells remains a challenge owing to their short sequence and most of them are expressed in cells at low levels. This project aims to develop new methods for highly sensitive in situ analysis of AD related miRNAs in living cells to solve the problem of insufficient sensitivity for miRNAs in situ analysis in living cells，based on our previous research of imaging and detection of miRNAs in living cells, combined with the utility of nanostructure assembly and biotechnology, as well as new achievements in areas of chemistry, material science and enzyme-free signal amplification technology, this project will design signal amplification probe with target-recognition properties, establish “on-off” type signal mode, then new methods and technologies for highly sensitive in situ real-time dynamic imaging of miRNAs in single living cells and convenient and highly sensitive in situ detection of miRNAs relative content in living cells will be proposed. This project will provide powerful methods and technologies for the intracellular miRNAs research, and contribute significantly to the diagnosis of AD。

广西百色是亚洲重要的铝工业基地，高铝暴露与阿尔茨海默病(AD)密切相关。miRNAs是潜在的疾病标志物和治疗靶点，它们在AD的发生发展过程中起着关键的调控作用，所以发展AD相关miRNAs活细胞内原位分析具有重要意义。由于序列短和绝大部分miRNAs在细胞中低表达，miRNAs在活细胞内的原位分析仍然是一个挑战。针对活细胞内miRNAs原位分析灵敏度不高的难题，本项目以发展AD相关miRNAs活细胞内高灵敏原位分析新方法为目标，在前期活细胞内miRNAs成像与检测的工作基础上，利用纳米组装与生物技术，结合化学、材料和无酶信号放大领域的新成就，设计特异性信号放大探针，构建“开-关”型信号模式，建立对单个活细胞内miRNAs实时动态荧光高灵敏原位成像和简便高灵敏的miRNAs相对含量检测分析的新技术和方法。本项目将为活细胞内miRNAs的研究提供有力的技术和方法，对AD的诊断也具有积极意义。

miRNAs等活性分子在预防阿尔茨海默病(AD)发生发展中起着重要作用，因此构建miRNAs等活性分子高灵敏测定方法在AD预警及相关机理研究中具有重要意义。高灵敏纳米生物分析方法的建立是进行miRNAs等活性分子功能研究的基础。基于纳米材料和“DNA信号放大”技术，利用纳米与分子生物学技术，本项目研究了系列纳米材料的制备、功能化及相关性能，探索了系列核酸介导的信号放大策略，提出了多种信号放大新思路，发展了系列生物传感新技术用于miRNAs等活性分子的高效传感，建立了系列高特异性、高灵敏度的miRNA等活性分子的检测新方法，实现了miRNAs的活细胞内原位信号放大分析检测，为活细胞内低丰度活性分子的原位研究提供新分析检测技术，为miRNA等活性分子的生物医学研究提供了重要的技术支持和理论参考，为精准医疗提供了新手段。项目执行期间，己在国际学术刊物上发表SCI论文13篇，协助培养博士研究生1名和硕士研究生3名，获得广西青年科技奖和广西科学技术奖(自然科学奖)三等奖各1项，参与获得广西科学技术奖(科技进步奖)一等奖1项。