目的 探索早期补充硫胺素对海马神经元细胞DNA甲基化及Aβ染毒的干预作用及机制。方法 新生大鼠海马神经元细胞（HNCs）体外培养,随机分为空白组、模型组及硫胺素低、中、高剂量组,进行不同剂量硫胺素干预3 d,HPLC法测定HNCs细胞DNA整体甲基化,质谱法检测CaMKIIα和GluR-2启动子甲基化,继续空白B27培养液培养至细胞成熟,观察硫胺素早期干预对HNCs成熟期Aβ1-42染毒后细胞增殖率（PR%）、细胞凋亡率（AR%）的影响。结果 ①与空白组比较,硫胺素低、中剂量组HNCs整体DNA甲基化水平均有所降低,中剂量组显著低于空白组（P<0.05）;②与空白组比较,硫胺素低剂量组CaMKIIα基因启动子CpG_3、CpG_11位点甲基化水平明显降低（P<0.05）,GluR-2基因CpG_5、CpG_23位点甲基化水平明显升高（P<0.05）;③Aβ染毒后,与空白组比较,模型组HNCs PR%显著下降、AR%明显升高（P<0.05）;与模型组比较,硫胺素干预各组Aβ染毒HNCs PR%明显升高（P<0.05）,高剂量组较低、中剂量组显著升高（P<0.05）;与模型组比较,硫胺素干预各组Aβ染毒HNCs AR%明显降低（P<0.05）,3个剂量组之间两两比较差异无统计学意义（P<0.05）。结论 硫胺素早期干预,能调节神经元细胞的甲基化水平,通过降低CaMKIIα启动子甲基化水平的表观遗传学机制,提高成熟期神经元细胞的抗Aβ神经毒性作用。
Abstract
Objective To explore the effects of early thiamine intervention on DNA methylation and intervention Aβ neurotoxicity in hippocampal neuron cells (HNCs). Methods HNCs from neonatal rats were cultured in vitro, and randomly divided into blank group, model group and low, medium and high dose groups of thiamine intervention. The HNCS were treated with different doses of thiamine for 3 days. The whole DNA methylation level of HNCs was determined by HPLC. The promoter methylation of CaMKIIα and GluR-2 genes was detected using MassARRAY. The cells were cultured in blank B27 medium until the cells matured. The effects of Aβ1-42 on the proliferation rate (PR%) and apoptosis rate (AR%) of HNCS were studied. Results (1) The DNA methylation level of HNCs early exposed to thiamine treatment was lower than the blank group,and the medium dose group showed a significant difference (P<0.05). (2) Compared with the blank group, the methylation level of CaMKIIαpromoter CpG 3,11 in the low dose thiamine group decreased significantly (P<0.05),and the methylation level of GluR-2 promoter CpG5, 23 increased significantly (P<0.05). (3) The treatment of Aβ1-42 caused a decreased cell proliferation rate and increased apoptosis rate. Compared with the model group, early thiamine intervention could increase proliferation rate. The high dose group was higher than the low and medium dose groups (P<0.05). Compared with the model group, early thiamine intervention could decrease apoptosis rate (P<0.05). There were no significant differences ( P>0.05) between different thiamine intervention groups. Conclusion Early thiamine intervention can regulate the methylation level of HNCs, and protect against the Aβneurotoxicity of mature hippocampal neuron cells through reducing the methylation level of CaMKIIαpromoter .
关键词
硫胺素 /
DNA甲基化 /
阿尔茨海默病
Key words
thiamine /
DNA methylation level /
Alzheimer’s disease
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基金
辽宁省科学事业公益研究基金计划项目（No.GY - 2017 - 0032）; 辽宁省高水平现代化高职院校和高水平特色专业群建设计划专项资金（辽教函[2017]798）
