DNA甲基化、去甲基化过程的电化学研究

DNA methylation and demethylation play important roles in many fundamental biological processes including transcription regulation, stem cell pluripotency, and cancer. Understanding the mechanisms of DNA methylation and demethylation is important in illustrating the epigenetic and genetic regulation of normal and disease development and could have potential therapeutic applications. The aim of this subject is to investigation of DNA methylation and demethylation process by electrochemical methods. We choose human colon cancer DNA as the researh object, using electrochemical method and in situ spectroscopic techniques, to develop electrochemical approachs for detection of the different pyrimidine bases, such as cytosine(C), 5-methylcytosine (5-mC) , 5-hydroxymethylcytosine(5-hmC), 5-carboxylcytosine (5-caC) and 5-hydroxymethyluracil (5-hmU), which were caused by methylation and demethylation. On this basis, we investigate DNA methylation and demethylation process, assay of protein (enzyme) activity and the kinetics character. In addition, we investigate the effects of temperature, solution pH and protein (enzyme) on the DNA methylation and demethylation process. The results obtained from the study of the project are significance in developing the novel DNA methylation and demethylation biosensor with high sensitivity and stability for the determination of the pyrimidine bases. Morover, the results are in favor of understanding the DNA methylation and demethylation process, providing important theoretical guidance for the people in diagnosis of disease and find the right treatment.

DNA甲基化、去甲基化在许多基本的生物过程，如转录调控、干细胞多能性、及癌症中扮演着重要的角色。本项目提出利用电化学方法研究DNA甲基化、去甲基化过程。以人类结肠癌DNA为研究对象，利用电化学方法结合原位光谱学技术，建立对DNA甲基化、去甲基化过程中产生的不同碱基，如胞嘧啶、5-甲基胞嘧啶、5-羟甲基胞嘧啶、5-羧基胞嘧啶的电化学检测方法。在此基础上，监测甲基化、去甲基化的过程，研究蛋白质（酶）的催化活性及不同机制下的动力学特征，考察各种因素如pH值、温度、蛋白质（酶）等对于其过程的影响。本项目的研究结果不仅对开发灵敏度高、稳定性好的DNA生物传感器具有重要的指导意义，并且有助于更深入地了解DNA甲基化、去甲基化的过程，从分子水平上解释某些疾病的致病机制，为疾病的诊断与治疗提供重要的理论指导。

DNA甲基化、去甲基化在许多基本的生物过程，如转录调控、干细胞多能性及癌症中扮演着重要的角色。本课题利用电化学、电致化学发光、荧光方法结合信号放大实现了对DNA甲基化水平、DNA甲基化转移酶（M.SssI以及DNMT1）活性的高灵敏检测，研究了几种抗癌药物对甲基转移酶活性的抑制作用，并对不同癌细胞内DNMT1的活性进行了检测和比较，其结果与商品化试剂盒测试结果保持一致。利用电化学、荧光方法研究了DNA去甲基化水平和DNA去甲基化酶活性。利用电化学方法结合DNA杂交链反应实现了对含有5-甲基胞嘧啶、5-羟甲基胞嘧啶的DNA的同时电化学检测。利用电致化学发光方法实现了对Hsp53 肿瘤抑制基因的甲基化水平和位置的有效检测。针对近几年的DNA甲基化分析的文章进行了总结，归纳，整理了一篇综述文章。其研究结果不仅有助于DNA甲基化、去甲基化的研究，而且为分析肿瘤细胞提供了有用的平台。其在DNA甲基化相关的临床诊断中具有很大的应用潜力，对发现新的抗癌药物方面具有潜在的应用价值。