DNA甲基化的同位素稀释质谱分析方法

DNA methylation detection is a promising test for early diagnosis of cancer as aberrant DNA methylation modification is an important mechanism of tumorigenesis. Recently, it is found that the positioning and quantitative analysis of DNA methylation in tumor diagnosis is most valuable, but the difficulties of the analysis is unable to take into account the efficiency，sensitivity and reliability at same time. The objective of this project is to establish a new method for positioning and quantitative analysis of DNA methylation by using isotope dilution-liquid chromatography tandem mass spectrometry (LC-MS/MS) combined with PCR amplification.The method including converted DNA by bisulfite, designed specific PCR primer for amplifing the DNA Candidate segment and analyzed cytosine and adenine by LC-MS, then caculated the DNA methylaion level. The innovation of the project is using accurate quantitative ability of isotope dilution mass spectrometry to solve the quantitative problem of DNA methylation of specific segment. Forthermore, we will validate the utility of our new method in patients with colorectal cancer. Specimens (biopsy samples, plasma, feces) from the cancer patients will be examined for DNA methylation profiles using the new method, aiming to identify highly sensitive and specific DNA methylation markers. The results of the project may have potential applications for early diagnosis and prognosis evaluation of colorectal cancer as well as for drug discovery. At the same time, it will promote application of mass spectrometry technology in biomedical field.

DNA甲基化的异常修饰是肿瘤发生、发展的重要机制，因而有望成为恶性肿瘤早期诊断的重要标志物。近来发现DNA甲基化的定位定量分析对肿瘤诊断极具价值，但现有方法难以兼顾灵敏、高效和可靠性。本项目拟运用同位素稀释质谱技术，结合PCR扩增手段，建立DNA甲基化的定位定量分析方法。该方法包括DNA的亚硫酸氢盐处理，设计PCR引物扩增候选区域DNA，通过LC-MS分析扩增产物中胞嘧啶和腺嘌呤的含量，计算该区域的DNA甲基化水平。本项目创新点在于运用同位素稀释质谱的精确定量能力解决DNA特定区域甲基化定量难题。在此基础上，以结直肠癌为研究对象，通过对其组织和血浆等样本的分析，寻找结直肠癌患者DNA甲基化的特点，期望发现特异性强和灵敏度高的DNA甲基化肿瘤标志物。该项目的研究对结直肠癌的早期诊断、预后判断和新药筛选等有潜在的应用价值，同时会推动质谱技术在生物医学领域的应用发展。

本项目在国家自然科学基金的资助下，开展了DNA甲基化的同位素稀释质谱分析方法研究。一是建立了基因组DNA甲基化及其氧化产物的LC-MS/MS分析方法，并应用于肿瘤组织、细胞中的甲基化、羟甲基化和羧甲基化水平，还探讨了n-3脂肪酸逆转肿瘤低甲基化的抗癌机制，以及AD小鼠中的DNA甲基化和羟甲基化改变。二是运用同位素稀释质谱的精确定量能力解决DNA特定区域甲基化定量难题，为特定基因区域DNA甲基化提供了一套新的定量方法，并筛选出CNRIP1、ALX4、RUNX3、FBN1、p16等多个基因，其甲基化水平在正常组织和结直肠癌组织中存在显著差异，能够很好地指示结直肠癌的发生发展，可作为结直肠癌临床诊断预后判断的潜在甲基化标志物。本项目的研究对结直肠癌的早期诊断、预后判断和新药筛选等有潜在的应用价值，同时也推动质谱技术在生物医学领域的应用发展。.基于上述研究成果，本项目已发表论文10篇，其中SCI论文6篇，而且仍有3篇SCI正在投稿中；培养硕士研究生7名，其中2人获得国家研究生奖学金，1人获得广东省优秀硕士学位论文。.本项目较好地执行了原定计划并完成了预期目标。