摘要
为研究DNA甲基化在帕金森病发病机制中的作用,本研究用环境毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)连续腹腔给药诱导小鼠帕金森病(Parkison's disease,PD)模型,应用ELISA检测小鼠黑质脑组织总体甲基化水平,应用实时荧光定量PCR方法检测DNA甲基转移酶表达水平,探讨MPTP诱导的小鼠PD模型黑质部位是否存在DNA甲基化异常.进一步应用甲基化DNA免疫共沉淀结合DNA甲基化芯片方法,构建MPTP诱导的小鼠PD模型黑质脑组织DNA甲基化谱,并寻找DNA甲基化修饰异常的PD相关基因对其进行验证.结果表明,模型组小鼠黑质脑组织DNA总体甲基化水平较对照组显著降低,Dnmt1的表达水平显著增高.利用DNA甲基化芯片在全基因组内筛选出甲基化差异修饰位点共48个,涉及44个基因,这些甲基化差异基因参与信号转导、分子转运、转录调控、发育、细胞分化、凋亡调控、氧化应激、蛋白质降解等生物学过程.在甲基化差异修饰基因中,对Uchl1基因及Arih2基因进行了甲基化水平以及表达水平的验证.结果表明,模型组小鼠黑质脑组织Uchl1启动子区域甲基化水平较对照组增高,m RNA及蛋白质表达水平降低,Arih2启动子区域甲基化水平较对照组降低,m RNA及蛋白质表达水平增高.实验结果进一步证实,DNA甲基化修饰异常在帕金森病发病机制中有重要作用,环境因素(如MPTP)可以通过改变DNA甲基化修饰参与帕金森病的发生发展.
The importance of DNA methylation in neurodegenerative diseases has been increasingly recognized.We explored role of DNA methylation in the pathogenesis of Parkinson＇s disease in mouse model induced by MPTP. The global DNA methylation levels of substantia nigra region were measured by ELISA Kit. Expression of Dnmt1 and Dnmt3 a were measured by Real-time PCR. Genome-wide profile was performed using methylated DNA immunoprecipitation microarray（Me DIP-Chip）. Methylation status of differential methylated gene was validated by bisulfate sequencing and expression of differential methylated gene was determined by Real-time PCR. We have demonstrated that global methylation level was significantly decreased while expression levels of Dnmt1 was significantly increased in substantia nigra region of MPTP-induced mouse model compared to saline controls. Genome-wide DNA methylation analysis detected 48 sites, involving 44 genes, with significantly altered DNA methylation. The abnormal-methylated genes involved in the biological processes concerning signal transduction, molecular transport, transcription modulation, development, cell differentiation, regulation of apoptosis, oxidation reduction and protein catabolism. The methylation levels of promoter region of Uchl1 in substantia nigra region of MPTP-treated mice were significantly higher than that in the saline controls, with significant decreased expression of m RNA and protein. The methylation levels of promoter region of Arih2 in substantia nigra region of MPTP-treated mice were significantly decreased than that in the saline controls, with significant increased expression of m RNA and protein. These results suggested that DNA methylation were altered in substantia nigra region of MPTP-induced PD model. DNA methylation may play important role in the pathogenesis of PD induced by environmental factors such as MPTP.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2015年第3期277-285,共9页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金国际(地区)合作与交流(81361120404)
国家自然科学基金青年基金(81401060)资助项目
国家重点基础研究发展计划(973)(2011CB510000)~~
