农药对基因组mC、hmC干扰的电致化学发光检测及其致癌性能的研究

It is important to detect cancer markers because it has the vital significance on clarifying the mechanism of cancer and improving the ability of human to intervene and prevent cancer. In this project, we focus on the development of new electrochemiluminescence method for assay of the expression level change of 5-methylcytosine and 5-hydroxymethylcytosine in genome DNA to investigate the pesticide carcinogenic mechanism. Firstly, we recombine the methyl binding domain protein, which can specifically recognize the 5-methylcytosine in double-stranded DNA，and modify the 5-hydroxymethylcytosine in DNA with amino compound. Based on methyl binding domain protein and amino modified 5-hydroxymethylcytosine coupling with new electroluminescent materials respectively, the new electrochemiluminescence method for detection of 5-methylcytosine and 5-hydroxymethylcytosine in genome DNA could be developed with high sensitivity and selectivity. Secondly, the possible interference factors of pesticide on DNA methylation are investigated based on the determination of the activity of DNA methyltransferase, and the relationship between oxidative stress system and the change of the amount of 5-methylcytosine and 5-hydroxymethylcytosine. Thirdly, under pesticide exposure condition, the expression level of 5-methylcytosine and 5-hydroxymethylcytosine, genetic damage and key enzyme activity in biological development process are systematacially investigated. Based on these results, the toxicological effect and pathogenic mechanisms of pesticide can be clarified. Through the investigation of this project, we could not only develop novel methods and techniques for the evaluation of toxicological and pathogenic effects of environmental pollutants, drug screening and food safety, but also provide new ideas for the investigation of carcinogenesis mechanism of environmental pollutants.

癌症标记物的检测，对揭示癌症发生的机理和增强人类阻止和干预癌症发生的能力具有重要的意义。本课题拟开发检测基因组内5-甲基胞嘧啶（mC）和5-羟甲基胞嘧啶（hmC）表达水平变化的电致化学发光新方法，从而探讨农药致癌机理的研究计划。首先以甲基结合域蛋白附着DNA链中的mC，以氨基化合物衍生DNA链中的hmC，再偶联电致化学发光新材料，开发灵敏度高、特异性强的mC和hmC检测的电致化学发光新方法。然后，在农药干扰体系中，考察DNA甲基转移酶活性、氧化应激系统对基因组中mC、hmC表达水平变化的影响，探索农药对DNA链中mC、hmC干扰的可能因素。进而通过对农药暴露下生物发育过程中mC和hmC表达水平的检测、基因损伤和关键酶活性的系统分析，阐明农药毒理作用及致癌机制。本研究不仅能为环境毒物毒理作用及致病效应的评价、药物筛选、食品安全等提供新的方法与技术，并且将为环境毒物致癌机理的研究提供新的思路。

癌症标记物的检测，对揭示癌症发生的机理和增强人类阻止和干预癌症发生的能力具有重要的意义。本项目针对基因组内DNA甲基化和羟甲基化表达水平检测方法缺乏，检测灵敏度不高，环境污染物和抗癌药物对基因组DNA甲基化和去甲基化干扰不明等关键科学问题，开展了下列研究工作，进行了Bi2S3纳米棒、BiOI纳米片、g-C3N4、介孔硅等多种纳米材料的制备，甲基结合域蛋白MBD的合成，考察了这些材料的光、电催化及电子传递性能。基于上述材料构建了多种电化学、光电化学和电致发光生物传感器，结合纳米材料扩增、MBD蛋白识别、生物素-亲和素识别、免疫识别和酶切、酶催化扩增等多种技术，开展了对DNA甲基转移酶活性的评价和5-羟甲基胞嘧啶（5-hmC）、N6-甲基腺苷（m6A）等的简单、灵敏和高选择性的检测，DNA甲基转移酶的最佳检测限可达0.0067 unit/mL（电化学法）、0.035 unit/mL（光电化学），5-hmC的最佳检测限可达1.43 pM（电化学法）、0.16 nM（光电化学法）和0.047 nM（电致发光法），m6A的最佳检测限可达2.57 pM（电化学法）、3.23 pM（光电化学法）。利用研制的传感器评价了常见的抗癌药物和环境激素对DNA甲基化水平及甲基转移酶活性的抑制作用，研究表明，多数药物对甲基转移酶活性的抑制作用均存在剂量-效应关系，环境激素毒死蜱、甲基对硫磷、莠去津和甲基吡噁磷明显抑制M.SssI甲基转移酶活性，甲基吡噁磷比莠去津对M.SssI甲基转移酶有更强的抑制性能，说明甲基吡噁磷对人有更高的致病风险，BPA和壬基酚对DNA的甲基化具有明显的促进作用，促进率随着浓度的增加而逐渐增大，说明了BPA和壬基酚均有一定的致癌作用。利用开发的方法对细胞中5-hmC的含量和血清中m6A的表达进行了检测，发现与正常乳腺细胞比较，5-hmC在乳腺癌细胞中的含量大量降低，比较了正常人、乳腺癌和胃癌病人血清中m6A的表达，表明乳腺癌和胃癌病人血清中m6A的量明显高于正常人的量，初步揭示了环境激素对癌症发生的影响，探讨了环境激素的致病机制。