基于FRET技术血清RNase A的检测及其在肿瘤诊断应用中的研究

项目名称： 基于FRET技术血清RNase A的检测及其在肿瘤诊断应用中的研究

项目编号： No.81502586

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 王晓霞

作者单位： 北京大学

项目金额： 16.5万元

中文摘要： 核糖核酸酶A（RNase A）是血清中一个重要组分，但目前仍没有一种可以灵敏定量检测血清中RNase A的方法。根据申请人所在实验室在小核酸降解方向研究中所揭示的RNase A对双链RNA降解的位点特异性，我们选取了具有血清特异性切割位点（CA/UG和UA/UA）的siRNA作为检测RNase A活性的特异性底物，设计了基于荧光共振能量转移（FRET）原理的RNase A活性检测方法。通过此方法检测小RNA分子是否被RNase A降解的状态，进一步可以检测RNase A活性，检测灵敏度可达10-8mg/ml。在对11种肿瘤303例病人血清以及128例健康人对照的初步研究中发现胃癌、结肠癌和肺癌中RNase A活性显著下降。本申请项目拟优化FRET方法，扩大样本量，进一步探讨血清中RNase A与肿瘤发生发展、分类和预后之间的关系，这将对RNase A作为一项肿瘤诊断标记物具有重要指导意义。

中文关键词： 荧光共振能量转移；核糖核酸酶A；肿瘤诊断

英文摘要： RNase A has been characterized for a long time, but methods have not been extensively explored to measure its activity accurately and robustly. In our preliminary study, we comprehensively studied the properties of double-stranded RNA as a new type of RNase A substrate, and revealed that serum RNase A predominantly cleave double-stranded RNA site-specifically at two dinucleotide sites(CA/UG and UA/UA). Using this as a guideline, we designed and characterized a FRET（Fluorescence Resonance Energy Transfer ）method to measure the site-specific activity of human serum RNase A. The dynamic range of the RNase A assay was from 10-8 to 0.1 mg/ml. Assessment of site-specific RNase A activity was carried out on the serum from 11 different type of cancers including 303 patients and 128 healthy controls using the new method. RNase A activity was found to be markedly reduced in patients with gastric cancer,colon cancer and lung cancer. In this study , we will further optimize this method to provide a comprehensive guideline for quantification of RNase A activity in human cancers and elucidate the relationship between RNase A and specific cancer types, thus demonstrate site-specific serum RNase A activity might serve as a novel biomarker to facilitate tumor diagnosis.

英文关键词： FRET;RNase A;tumor diagnosis