中文摘要：

目的 探讨EIF2B3突变的少突胶质细胞是否存在对内质网应激（ERS）的不耐受,检测未折叠蛋白反应（UPR）通路的变化,为了解白质消融性白质脑病（VWM）的发病机制提供线索.方法 本研究利用转染EIF2B3野生型或突变型c.1037T＞C全长cDNA慢病毒载体的人类少突胶质细胞系,Thapsigargin对其进行ERS诱导后,通过细胞增殖-毒性检测试剂盒实验反映细胞增殖活力,检测凋亡相关分子Caspase-3剪切体的表达反映细胞凋亡水平,来验证野生与突变细胞对ERS的耐受性,并比较二者在基础状态及ERS诱导后不同时间点UPR激酶样内质网激酶（UPR-PERK）通路各分子指标的变化.结果 1.EIF2B3突变型少突胶质细胞对ERS不耐受,表现为ERS刺激后凋亡的增加和活力的显著下降.2.EIF2B3突变型少突胶质细胞存在UPR通路的过度激活.3.基础状态下UPR-PERK通路分子磷酸化α亚基的真核翻译起始因子2（P-eIF2α）、激活转录因子4、CCAAT增强子结合蛋白（CHOP）和生长抑制DNA损伤基因34（GADD34）的水平不同程度高于野生组.4.ERS刺激24h后,凋亡相关的UPR分子（CHOP和GADD34）和抑制蛋白翻译的P-eIF2α在野生型细胞中表达下降,而在突变细胞中持续高表达.结论 EIF2B3突变的人少突胶质细胞对ERS不耐受,这种不耐受与突变细胞存在UPR-PERK通路的过度持续激活,引起凋亡性的细胞死亡相关.减轻突变细胞的内质网负荷或稳定UPR的过度激活有望对疾病进展起到干预作用.

英文摘要：

Objective Intolerance to endoplasmic reticulum stress （ERS） and biomarkers in unfolded protein response （UPR） were measured,which provided clues to the investigation of potential pathogenesis.Methods Human oligodendrocytes cell lines transfected with full length EIF2B3 cDNA of the wild type or c.1037T 〉 C,were used as the cell model.Thapsigargin induced ERS condition.Cleaved Caspase-3 and cell viability assays （cell counting kit-8） were applied to reflect ERS tolerance between the wild type and mutant oligodendrocytes under baseline （0 h） or different time points after ERS induction.The components in UPR pancreatic ER kinase-like ER kinase（UPR-PERK） pathway were also measured under baseline condition and after ERS induction.Results （1） Oligodendrocytes transfected with the mutant EIF2B3 showed less tolerance to ERS than the wild type,with declined cell viability and increased apoptosis rates.（2）UPR pathway was over activated in human oligodendrocyte cell line transfected with EIF2B3 mutation.（3） Western blot showed UPR biomarkers of phosphorylated eukaryotic translation initiation factor 2α （P-eIF2α）,activating transcription factor 4,CCAAT enhancer-binding homologous protein （CHOP） and growth arrest and DNA damage inducible gene 34 （GADD34） were expressed at a higher level in the mutant cells under basal condition.（4）The expression of apoptosis related UPR factors （CHOP,GADD34） and P-eIF2α stayed at a higher level from 24 to 72 hours with the time of ERS stimulation in the mutant cells,but decreased in the wild type group.Conclusions Oligodendrocytes with EIF2B3 were less tolerable to ERS.The ERS susceptibility is related to UPR mediated apoptotic cell death.The relief of the endoplasmic reticulum burden and stabilization of the over-activated UPR are the potential approaches for the delay of the progressive aggravation of the disease.