摘要
目的观察百草枯(PQ)对人神经干细胞增殖的影响,探讨PQ基于DNA甲基化途径调控人神经干细胞增殖的作用机制.方法以永生化的人神经干细胞系(ReNcell CX)为体外模型,通过间接免疫荧光法检测巢蛋白(Nestin)、β-微管蛋白Ⅲ(β-tubulin Ⅲ)和胶质纤维酸性蛋白(GFAP)的表达,评估神经干细胞的自我更新和分化潜能;以终浓度0、5、25、50、100 μmol/L PQ作用细胞24h,以及50μmol/L PQ分别作用细胞6、12、24、48 h,四甲基偶氮唑盐(MTT)检测细胞存活率;应用终浓度0、25、50、100 μmol/L PQ处理细胞24 h,采用Sox2/Brdu、Nestin/Brdu免疫荧光双标记法检测神经干细胞的增殖能力;采用DNA甲基化定量试剂盒检测细胞总甲基化水平;采用实时荧光定量PCR(RT-PCR)和蛋白质免疫印迹法(Western blot)检测DNA甲基转移酶(Dnmt1、Dnmt3a、Dnmt3b)的转录和表达变化.结果增殖期的干细胞能够大量表达Nestin蛋白,经诱导分化后,可获得表达相应特异性标志蛋白的神经元(β-tublin Ⅲ)和星形胶质细胞(GFAP);MTT显示,与对照组比较,PQ呈剂量依赖性和时间依赖性抑制ReNcell CX人神经干细胞的活力;免疫荧光双标记染色显示,Brdu和Sox2均呈双阳性表达,各个染毒组的Brdu+/Sox2+双标阳性率随PQ浓度增加逐渐降低(P<0.05);Brdu+/Nestin+双标阳性率也随PQ浓度增加而逐渐降低,其中50、100 μmol/L染毒组的双标阳性率较对照组明显降低,差异有统计学意义(P<0.05);DNA总甲基化水平检测结果显示,与对照组比较,25、50、100 μmol/L PQ染毒组细胞的总甲基化水平降低,差异有统计学意义(P<0.05);Western blot结果显示,与对照组比较,50、100 μmol/L PQ染毒组的Dnmt1、Dnmt3a蛋白表达水平明显降低,差异有统计学意义(P<0.05);50、100 μmol/L染毒组的Dnmt3b蛋白表达水平明显升高,差异有统计学意义(P<0.05);RT-PCR结果显示,与对照组比较,25、50、100 μmol/L PQ染毒组的Dnmt1、Dnmt3a基因表达水平降低,50、100 μmol/L PQ染毒组Dnmt3b基因表达水平明显升高(P<0.05).结论 25、50和100 μmol/L PQ可能通过降低DNA甲基化转移酶(Dnmt1、Dnmt3a)的蛋白表达和转录水平,抑制DNA甲基化反应的发生,进而抑制人神经干细胞的增殖.
Objective To investigate the effects of Paraquat on neural stem cell proliferation in vitro and explore the its mechanism based on DNA methylation pathway.Methods Nestin,β-tubulin Ⅲ,and glial fibrillary acidic protein (GFAP) were detected by indirect immunofluorescence assay to evaluate self renewal and differentiation potentia of ReNcell CX human neural stem.The cells were treated with terminal concentrations of0,5,25,50,and 100μmol/L PQ for 24 hours,and the cells were induced by 50 μmol/L PQ for different time (6,12,24,48 h).Cell viability was determined by MTT assay.The proliferation of neural stem cells was evaluated using Sox2/Brdu and Nestin/Brdu double immunofluorescence staining.The global DNA methylation level was assayed by MethyflashTM methylated DNA Quantification kit.The expression levels of Dnmts mRNA and protein were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot,respectively.Results Immunofluorescence showed that nestin was primarily expressed in proliferative neural stem cell and peotein biomarkers (β-tubulin Ⅲ,GFAP) for neuron and astrocyte were expressed in differentiated cells.MTT assay showed PQ induced cell survival rate decrease in a time and dose dependent manner.Double immunfluorescence staining of cells showed colocalization of Sox2 and Brdu.The percentage of Brdu/Sox2 positive cells was significantly lower in the PQ-exposed (25,50,100μmol/L PQ treatment) groups compared to control (P<0.05);Meanwhile,The percentage of Brdu/Nestin positive cells was also significantly lower in the PQ-exposed (50,100xmol/L PQ treatment) groups compared to control (P<0.05).The results of global DNA methylation revealed a significant decrease in PQ-exposed groups (P<0.05).Western blot showed that compared with control group,the protein and mRNA levels of Dnmtl,Dnmt3a in PQ-exposed group were significantly decreased (P<0.05),but there was a significant increase in expression level of Dnmt3b in 50,100 μmol/L PQ-treated group(P<0.05).Conclusion Paraquat could inhibite the proliferation of human neural stem cells through reducing the level of DNA methylation reaction by suppressing the protein expression and transcription of DNA methylated transferase (Dnmts).
作者
黄敏
黄楠楠
李颖颖
武坷鑫
郭慕真
朱晨笛
Huang Min;Huang Nannan;Li Yingying;Wu Kexin;Guo Muzhen;Zhu Chendi(School of Public Health, Ningxia Medical University, Yinchuan 750004, China)
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2019年第3期161-168,共8页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
国家自然科学基金项目(81560538)
宁夏高等学校一流学科建设(公共卫生与预防医学学科)资助项目(NXYLXK2017B08)
大学生创新/创业计划项目(NXCX2017101).
