BAT3过表达缓解由PrP106-126毒性多肽诱导的神经元凋亡  

Overexpression of BAT3 Alleviates Prion Protein Fragment PrP106-126-Induced Neuronal Apoptosis

作　　者：宋志琦[1] 赵文杰[2] 杨利峰[1] 王运盛[1] 朱婷[1] 程广宇[1] 周向梅[1] 赵德明[1] 

机构地区：[1]中国农业大学动物医学院国家动物海绵状脑病实验室,北京100193 [2]河南农业大学牧医工程学院,郑州450000

出　　处：《畜牧兽医学报》2015年第5期841-848,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31272532)

摘　　要：拟检测BAT3蛋白过表达对由PrP106-126毒性多肽引起的神经元凋亡现象的影响。首先通过免疫荧光、CCK8细胞活性检测试剂盒和免疫印迹检测带FITC标签的PrP106-126-FITC毒性多肽的生物学毒性。分别用PrP106-126-FITC和PrP106-126神经毒性多肽处理原代神经元和Neuro2a细胞,通过免疫荧光技术和免疫印迹方法分别检测受刺激后的原代神经元和Neuro2a细胞中BAT3表达的变化情况。通过脂质体转染方法将已经构建好的pcDNA-3.1-HA-BAT3质粒转染进入原代神经元,用CCK8检测细胞活性;以同样方式将质粒转染进入小鼠神经瘤细胞系Neuro2a,用TUNEL凋亡检测试剂盒分析经BAT3转染或对照组的N2a细胞的凋亡水平;Western blot检测PrP106-126神经毒性多肽处理前后,胞质内细胞色素C和Bcl-2的变化,进一步了解BAT3在神经元凋亡中发挥作用的机制。结果显示,PrP106-126-FITC和PrP106-126神经毒性多肽具有相似的神经毒性。受到多肽刺激之后,无论在原代神经元还是传代细胞系Neuro2a中,BAT3都发生核输出现象,BAT3从细胞核转移进入细胞质。BAT3的过表达使受到PrP106-126毒性多肽刺激的神经元的细胞活性有所提高,Neuro2a的细胞凋亡现象减轻,同时抑制了由PrP106-126毒性多肽引起的细胞色素C的过度释放并且维持细胞内抗凋亡蛋白Bcl-2的稳定。BAT3蛋白的表达缓解PrP106-126毒性多肽对神经细胞造成的凋亡现象,为进一步阐释该蛋白质对朊病毒引起的神经元损失的治疗作用机制提供了依据。The objective of this study was to investigate the interactions between BAT3 and prion protein and the potential role of BAT3 in PrP106-126-induced apoptosis.Immunofluorescence,the CCK-8assay kit and western blotting were used to test the cytotoxicity of PrP106-126-FITC.After the stimulation of PrP106-126-FITC or PrP106-126,the location of BAT3 was tested by immunofluorescence and western blotting in the neurons.BAT3 was overexpressed in Neuro2 aor pri-mary neuronal cells by transfection with pcDNA3.1-HA-BAT3 expression plasmids.The effect of BAT3 on PrP106-126-induced cytotoxicity and apoptosis were detected by the CCK-8assay and TUNEL assay.The expression of cytochrome c and Bcl-2were examined by western blotting.Results were as follows:PrP106-126-FITC has similar cytotoxicity with PrP106-126.BAT3 interacted with prion protein and enhanced PrP expression.After PrP106-126 peptide treated,BAT3 was transported from the nucleus to cytoplasm,increased cell viability and protected neurons from PrP106-126-induced apoptosis through stabilizing the level of Bcl-2protein and inhibiting the release of cytochrome c to cytoplasm.Our present data showed a novel molecular mechanism of PrP106-126-induced apoptotic process regulation through the overexpression of BAT3,which may be important for the basic regulatory mechanism of neuron survival in prion diseases and associated neurodegenerative diseases in vivo.

关 键 词：BAT3 PrP106-126毒性多肽 神经凋亡 传染性海绵状脑病 原代神经元 

分 类 号：R742.9[医药卫生—神经病学与精神病学]