人源酸敏感离子通道1a介导酸引起细胞凋亡的研究  

Human acid-sensing ion channel 1a mediates worsen acid-induced cell apoptosis

作　　者：徐媛媛 李文娜 翟玉荣 莫镇涛 李意奇 李俊明 肖璐 李彩兰 Xu Yuanyuan;Li Wenna;Zhai Yurong;Mo Zhentao;Li Yiqi;Li Junming;Xiao Lu;Li Cailan(Zhuhai Campus of Zunyi Medical University,Zhuhai Guangdong 519041,China)

机构地区：[1]遵义医科大学珠海校区

出　　处：《遵义医科大学学报》2019年第6期618-622,共5页Journal of Zunyi Medical University

基　　金：珠海市优势学科研究计划项目;遵义医科大学博士启动基金资助项目(NO:F-953)

摘　　要：目的比较人与小鼠来源酸敏感离子通道1a(ASIC1a)介导酸引起的细胞凋亡。方法对COS7细胞以及培养的原代皮层锥体神经元转染人或小鼠源ASIC1a质粒,COS7细胞经过pH7.4/pH6.0处理2 h,神经元经过pH7.4/pH6.0处理1 h,加入PI染色,统计PI阳性细胞比率;对培养的CHO细胞转染人和小鼠源ASIC1a质粒,经过pH7.4/pH6.0/pH6.0+PcTx-1处理2 h,通过荧光显微镜拍照,统计细胞死亡率,通过MTT实验检测细胞活力;采用生物素标记、亲和素沉淀分离ASIC1a膜蛋白,通过免疫印迹实验定量分析ASIC1a膜蛋白。结果与转染小鼠源ASIC1a的COS7细胞和神经元相比,转染人源ASIC1a的COS7细胞和神经元,由酸引起的细胞PI阳性细胞比率更大,细胞死亡率更高,细胞活力更低,且ASIC1a特异性阻断剂PcTx-1能显著逆转由酸引起的细胞凋亡,人源ASIC1a在细胞膜表达更多。结论人源ASIC1a比小鼠源ASIC1a介导酸引起的细胞凋亡更重。Objective To compare with the acid-induced cell apoptosis mediated by human ASIC1a and mouse ASIC1a.Methods The COS7 cells/primary cerebral cortex pyramidal neurons/CHO cells were transfected with human or mouse ASIC1a plasma,the COS7 cells were treated with solutions buffered at pH7.4 or pH6.0 for 2 h,the neurons were treated with solutions buffered at pH7.4 or pH6.0 for 1 h,both COS7 Cells and neurons were stained as red by propidium iodide(PI),We quantified the percentage of PI positive cells/green cells.The CHO cells were treated with pH7.4/pH6.0/pH6.0+PcTx-1 solutions buffered for 2 h,We counted the number of remaining GFP-positive cells with fluorescence microscope and We quantified the cell viability in MTT.We further investigated the ASIC1a surface expression with biotinylation,neutravidin pull down and Western blot experiments.Results Compared with mouse ASIC1a,human ASIC1a exhibited a higher acid-activated percentage of PI positive in both COS7 cells and neurons.Human ASIC1a exhibited a larger cell death rate and a lower cell viability in CHO cells.Human ASIC1a also exhibited a higher surface expression.Conclusion Human ASIC1a mediates worsen acid-induced cell apoptosis as compared with mouse ASIC1a.

关 键 词：人源ASIC1a 小鼠源ASIC1a 酸 凋亡 膜表达 

分 类 号：R965[医药卫生—药理学]