摘要目的 评价大鼠脊髓神经元氧糖缺失-复氧复糖损伤时10-11易位蛋白3(TET3)诱导DNA去甲基化在甲烷上调核转录因子E2相关因子2(Nrf2)表达中的作用.方法 原代培养大鼠脊髓神经元,以1×105个∕ml密度接种于6孔板,采用随机数字表法分为5组(n=48):正常对照组(C组)、氧糖缺失-复氧复糖组(OGD∕R组)、甲烷组(M组)、甲烷+TET3-siRNA组(M+siTET3组)和甲烷+阴性siRNA组(M+siCon组).采用无糖-无血清Earle平衡盐液,在5％CO2-95％N2缺氧培养箱中孵育2h,然后正常培养的方法制备氧糖缺失-复氧复糖损伤模型.M组于复氧复糖时在培养液中加入200μl甲烷饱和生理盐水(甲烷终浓度1.8 mmol∕L);M+siTET3组和M+siCon组于复氧复糖前24 h时分别加入100 pmol∕L TET3-siRNA和100 pmol∕L阴性siRNA进行转染.复氧复糖12 h时,测定神经元存活率、LDH漏出率、神经元凋亡率,采用实时荧光定量PCR法检测TET3和Nrf2的mRNA表达,采用Western blot法测定核蛋白Nrf2和TET3表达水平;采用ELISA法测定神经元SOD、过氧化氢酶(CAT)和MDA含量.提取神经元DNA,采用ELISA法确定DNA甲基化率、羟甲基化率,采用实时荧光甲基化特异性PCR法检测Nrf2基因启动子CpG岛甲基化水平.结果 与C组比较,OGD∕R组神经元存活率降低,LDH释放率和凋亡率升高(P<0.01);与OGD∕R组比较,M组神经元存活率升高,LDH释放率和凋亡率降低,TET3及其mRNA、Nrf2及其mRNA表达上调,DNA羟甲基化率、SOD和CAT含量升高,DNA及Nrf2启动子甲基化率、MDA含量降低(P<0.05或0.01);与M组比较,M+siTET3组神经元存活率降低,LDH释放率和凋亡率升高,TET3及其mRNA、Nrf2及其mRNA表达下调,DNA羟甲基化率、SOD和CAT含量降低,DNA及Nrf2启动子甲基化率、MDA含量升高(P<0.05或0.01),M+siCon组上述指标比较差异无统计学意义(P>0.05).结论 大鼠脊髓神经元氧糖缺失-复氧复糖损伤时甲烷上调Nrf2表达的机制与激活TET3,促进Nrf2启动子区DNA去甲基化有关.

abstractsObjective To evaluate the role of TET3-induced DNA demethylation in methane-in-duced up-regulation of nuclear factor-erythroid 2-related factor 2 ( Nrf2) expression in rat spinal cord neu-rons subjected to oxygen-glucose deprivation and restoration ( OGD∕R) injury. Methods The primarily cultured spinal cord neurons of rats were seeded in 6-well plates at a density of 1×105 cells∕ml and divided into 5 groups ( n=48 each) using a random number table method: control group ( group C) , group OGD∕R, methane group (group M), methane plus TET3-siRNA group (group M+siTET3) and methane plus negative siRNA group (group M+siCon). The medium was replaced with glucose- and serum-free Earle's salt solution, and the neurons were exposed to 5％ CO2-95％N2 in an incubator for 2 h followed by routine culture to establish the model of OGD∕R. In group M, 200μl methane-saturated saline ( final concentration of methane 1. 8 mmol∕L) was added at oxygen-glucose restoration. TET3-siRNA 100 pmol∕L and negative siRNA 100 pmol∕L were added at 24 h before oxygen-glucose restoration to perform transfection in M+siTET3 and M+siCon groups, respectively. At 12 h of oxygen-glucose restoration, the neuronal survival rate, release rate of lactic dehydrogenase ( LDH) and apoptotic rate of neurons were measured, and the ex-pression of TET3 and Nrf2 protein and mRNA was detected by Western blot and fluorescent quantitative re-al-time polymerase chain reaction, respectively, and contents of superoxide dismutase (SOD), catalase ( CAT) and malonaldehyde ( MDA) were measured by enzyme-linked immunosorbent assay. Neuronal DNA was extracted for determination of methylation and hydroxymethylation rates of DNA ( by enzyme-linked im-munosorbent assay) and methylation of CpG island in Nrf2 gene promoter ( by fluorescent real-time methyla-tion specific polymerase chain reaction). Results Compared with group C, the survival rate of neurons was significantly decreased, the release rate of LDH and apoptotic rate were increased in group OGD∕R ( P<0. 01) . Compared with OGD∕R, the survival rate of neurons was significantly increased, the release rate of LDH and apoptotic rate were decreased, the expression of TET3 and Nrf2 protein and mRNA was up-regula-ted, DNA hydroxymethylation rate and contents of SOD and CAT were increased, and the DNA and Nrf2 promoter methylation rates and MDA content were decreased in group M ( P<0. 05 or 0. 01) . Compared with group M, the neuronal survival rate was significantly decreased, the release rate of LDH and apoptotic rate were increased, the expression of TET3 and Nrf2 protein and mRNA was down-regulated, the DNA hydroxymethylation rate and contents of SOD and CAT were decreased, and the DNA and Nrf2 promoter methylation rates and MDA content were increased in group M+siTET3 ( P<0. 05 or 0. 01) , and no signifi-cant change was found in the parameters mentioned above in group M+siCon ( P>0. 05) . Conclusion The mechanism by which methane up-regulates Nrf2 expression in rat spinal cord neurons subjected to OGD∕R injury is related to activating TET3 and promoting DNA demethylation in Nrf2 promoter.