MANF对内质网应激诱导的细胞凋亡的保护作用

发布时间：2018-03-29 05:27

本文选题：中脑星形胶质细胞源性神经营养因子　切入点：转染　出处：《安徽医科大学学报》2013年11期

【摘要】：目的构建中脑星形胶质细胞源性神经营养因子(MANF)基因真核表达质粒并转染小鼠神经瘤细胞系(N2A),建立稳定表达MANF的细胞系,并观察MANF对内质网(ER)应激诱导细胞凋亡的影响。方法将MANF基因克隆至pEGFP-C2载体中,经酶切、聚合酶链式反应(PCR)、测序检测其构建的正确性,脂质体转染法将重组质粒转染入N2A细胞,经G418(500μg/ml)连续筛选,获得稳定表达MANF的神经细胞系。细胞免疫荧光法观察MANF的定位,蛋白质印迹法(Western blot)和反转录PCR法(RT-PCR)鉴定稳转细胞系中MANF的蛋白和基因表达水平。在稳定转染细胞系中,分别用4μg/ml衣霉素(TM)处理16 h和10μmol/Lβ淀粉样蛋白(Aβ)处理24 h诱导ER应激,Western blot法检测CCAAT增强子结合蛋白同源蛋白(CHOP)的表达和Caspase-3的激活情况。结果构建的pEGFP-C2-MANF质粒经酶切测序等鉴定确定构建成功,经G418抗性筛选出稳定转染MANF的N2A细胞系,RT-PCR、Western blot和细胞免疫荧光法证明稳转细胞中MANF的基因和蛋白水平均高表达,并且MANF的细胞定位准确。诱导ER应激后,MANF能显著抑制CHOP的表达和Caspase-3的激活。结论成功建立稳定表达MANF的神经细胞系,并且发现MANF可以抑制ER应激诱导的神经细胞凋亡。
[Abstract]:Objective to construct the eukaryotic expression plasmid of mesencephalic astrocyte-derived neurotrophic factor (MANF-) gene and transfect it into mouse neuroma cell line (N2AN), and to establish a cell line stably expressing MANF. To observe the effect of MANF on the apoptosis induced by endoplasmic reticulum (ER) stress. Methods the MANF gene was cloned into pEGFP-C2 vector, then digested by enzyme, polymerase chain reaction (PCR) and sequenced. The recombinant plasmid was transfected into N2A cells by liposome transfection. After continuous screening by G418G / ml, the stable expression of MANF in neural cell lines was obtained. The localization of MANF was observed by immunofluorescence assay. Western blotting and reverse transcription PCR assay were used to identify the protein and gene expression of MANF in stable transfected cell lines. The expression of CCAAT enhancer binding protein homologous protein (Caspase-3) and the activation of Caspase-3 were detected by ER stress Western blot method after treatment with 4 渭 g/ml and 10 渭 mol/L 尾 amyloid A 尾 for 16 h and 10 渭 mol/L 尾 -amyloid A 尾 for 24 h, respectively. Results the constructed pEGFP-C2-MANF plasmid was digested by enzyme. Sequence and other appraisals determined that the construction was successful, The stable MANF transfected N2A cell line was screened by G418 resistance. The expression of MANF gene and protein in the transfected N2A cell line was confirmed by Western blot and immunofluorescence assay. After inducing ER stress, the expression of CHOP and the activation of Caspase-3 were significantly inhibited by MANF. Conclusion the stable expression of MANF was successfully established and the apoptosis of neurons induced by ER stress was inhibited by MANF.
【作者单位】：安徽医科大学药学院;安徽医科大学生物药物研究所;安徽医科大学基础医学院;
【基金】：国家自然科学基金(编号:91129729、81173074) 教育部博士点基金(博导类)(编号:20113420110004)
【分类号】：R346

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