脊髓背角DNA甲基化介导NR2B基因表达上调参与疼痛慢性化的机制研究

Up-regulation of NMDA receptor 2B (NR2B)gene expression in the spinal dorsal horn is an important mechanism in chronicity of pain, but the underlying mechanism is still unclear. Recent studies found that change of DNA methylation is closely related to the increase of NR2B expression. Our preliminary data showed that down-regulated global DNA methylation was clearly detected in lumbar enlargement of spinal dorsal horn in a rat pain model, while NR2B expression in neurons was also increased. Together with other studies, we thus hypothesized that the change of DNA methylation in spinal dorsal horn induces increased expression of NR2B and contributes to the chronicity of pain. Thus in our project, based on chronic pain model of spinal nerve ligation, and by using a combination of the molecular biological, behavioral, morphological, and electrophysiological methods, we plan to observe the global DNA methylation level in the spinal dorsal horn, the expressional changes of DNA methyltransferase (DNMT) and methyl-CpG binding protein 2 (MeCP2) ,NR2B. We detect a large number of CpG sites within NR2B gene promoter region and related transcription factors which are directly impacted by DNA methylation. Then we evaluate the effects of DNA methylation substrate on pain behavior, expressional change of NR2B and relational analysis for the methylation level of NR2B gene promoter region. Further probe the regulating effect of DNA methylation on the expression of NR2B and related physiological consequences. We finally also analyze the upstream mechanism of changes in DNA methylation. Our proposed study may be helpful for further disclosing the mechanism of the chronicity of pain from DNA methylation role and may provide a novel strategies for chronic pain treatment.

脊髓背角NMDA受体亚单位NR2B基因表达的升高是疼痛转向慢性化的重要机制，但发生机理不清。新近研究发现NR2B表达的升高与DNA甲基化改变密切相关。我们前期运用大鼠痛模型观察到在疼痛慢性化阶段腰膨大脊髓背角总DNA甲基化水平降低，但神经元内NR2B的表达有所升高。结合他人研究，我们推测脊髓背角DNA甲基化能够调控NR2B表达而参与疼痛慢性化。本课题拟在大鼠脊神经部分结扎模型上综合运用分子生物学、行为学、形态学和电生理学等方法，检测痛状态下脊髓背角总DNA甲基化水平、甲基化转移酶和连接蛋白、NR2B的表达变化，检测NR2B启动子区CpG的甲基化位点和相关转录因子；观察给予甲基化底物对痛行为、NR2B和启动子区甲基化水平的影响，探讨其对NR2B表达的调控作用和相关的生理效应，最后分析导致DNA甲基化改变的上游分子机制。本项目将从DNA甲基化角度阐明疼痛慢性化的机制，为慢性痛的治疗提供新策略。

脊髓背角NMDA受体亚单位NR2B基因表达的升高是疼痛转向慢性化的重要机制，但发生机理不清。本项目综合运用行为学、形态学和分子生物学技术从表观遗传学角度集中探究了慢性痛状态下脊髓背角内NR2B基因表达发生改变的分子基础。主要结果如下：（1）小鼠后足底注射CFA构建炎性痛模型，7 d时处死动物。免疫组化结果显示：CFA组小鼠脊髓背角内NR2B免疫阳性产物明显多于正常对照组，同时实时定量PCR结果也提示，7 d时CFA组小鼠脊髓背角腰膨大节段NR2B mRNA的表达量与正常组比较增加了大约38.6% (P<0.05)。这些结果提示炎性痛后小鼠脊髓背角内NR2B表达的上调极有可能在小鼠慢性痛的发生中具有重要作用；（2）CFA模型成功之后在不同时间点对小鼠脊髓背角总体基因DNA甲基化水平进行测定，结果显示：随着时间的延长DNA甲基化水平逐渐升高，与正常组相比较，从第7 d开始实验组小鼠DNA甲基化水平已明显有所升高（P<0.05）。（3）利用重亚硫酸氢盐转换后PCR的方法，对NR2B基因启动子区，即转录起始位点上游约2000 bp及转录起始位点后约1500 bp内共3500 bp DNA序列中CpG位点甲基化水平进行检测，结果显示：NR2B基因启动子区发生去甲基化的区域集中在转录起始位点上游1300 bp到1200 bp之前的6个CpG位点处，而在其它区域未观察到明显的甲基化或去甲基化改变。本项目为从DNA甲基化角度阐明疼痛慢性化的机制提供了一定的理论依据。