摘要
目的·探索C9ORF72肌萎缩侧索硬化/额颞叶痴呆(amyotrophic lateral sclerosis/frontotemporal dementia,ALS/FTD)相关的多聚甘氨酸-精氨酸(poly-glycine-arginine,poly-GR)对线粒体形态及功能的影响,并分析二甲双胍对由poly-GR诱导的线粒体损伤的修复作用及其可能的机制。方法·采用慢病毒感染法分别构建能够稳定表达50个重复甘氨酸-精氨酸序列[(glycine-arginine)50,(GR)_(50)]和绿色荧光蛋白(green fluorescent protein,GFP)的SK-N-SH细胞,即(GR)_(50)-SK细胞株和GFP CTRL-SK细胞株。采用蛋白质印迹法(Western blotting)验证已构建细胞中(GR)_(50)蛋白的表达水平,并采用荧光显微镜观察GFP CTRL-SK细胞的GFP表达。采用碘化丙啶(propidium iodine,PI)染色分别检测(GR)_(50)-SK、GFP CTRL-SK细胞的凋亡水平。利用免疫荧光(immunofluorescence,IF)染色分析(GR)_(50)蛋白在细胞内的定位情况。采用超氧化物指示剂MitoSOX Red分别对(GR)_(50)-SK、GFP CTRL-SK细胞的氧自由基进行染色,并利用荧光显微镜观察红色荧光强度以评估线粒体活性氧水平的变化。采用透射电镜分别观察(GR)_(50)-SK、GFP CTRL-SK细胞的线粒体形态。采用Western blotting检测(GR)_(50)-SK、GFP CTRL-SK细胞的蛋白激酶B(protein kinase B,PKB,又称AKT)及其磷酸化水平。利用SC79激活(GR)_(50)-SK细胞中的AKT,并采用MitoSOX Red染色及PI染色实验分析AKT磷酸化后细胞的线粒体活性氧水平及凋亡水平。利用二甲双胍处理(GR)_(50)-SK、GFP CTRL-SK细胞,随后通过上述方法以及ATP检测试剂盒检测细胞的凋亡水平、线粒体活性氧水平、线粒体形态、AKT及其磷酸化水平、ATP浓度情况。结果·Western blotting结果提示(GR)_(50)-SK细胞构建成功,荧光显微镜的观察结果显示GFP CTRL-SK细胞构建成功。PI染色结果显示,(GR)_(50)-SK细胞较GFP CTRL-SK细胞的凋亡水平更高(P=0.016)。IF结果提示,(GR)_(50)-SK细胞中(GR)_(50)蛋白与线粒体存在部分共定位。�
Objective·To investigate the effect of C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia(ALS/FTD)-related poly-glycine-arginine(poly-GR)on mitochondrial morphology and function,and analyze the rescue effect of metformin on mitochondrial damage induced by poly-GR and its underlying mechanism.Methods·SK-N-SH cells stably overexpressing 50 repeated glycine-arginine sequences[(GR)_(50)]or green fluorescent protein(GFP)were constructed by lentivirus infection,which were respectively named as(GR)_(50)-SK cell line and GFP CTRL-SK cell line.(GR)_(50) expression in(GR)_(50)-SK cells was verified by Western blotting.GFP expression in GFP GTRL-SK cells was observed by fluorescence microscope.Propidium iodide(PI)staining was used to detect the apoptosis levels of(GR)_(50)-SK and GFP CTRL-SK cells.Immunofluorescence(IF)staining was performed to determine the subcellular location of(GR)_(50).Reactive oxygen species(ROS)level of mitochondria was evaluated by staining cells with MitoSOX Red followed by observing the intensity of red fluorescence under fluorescence microscope.The mitochondrial morphology of(GR)_(50)-SK and GFP CTRL-SK cells was observed by transmission electron microscopy.Western blotting was used to detect protein kinase B(PKB,also known as AKT)and its phosphorylation levels in(GR)_(50)-SK and GFP CTRL-SK cells.SC79 was used to activate AKT in(GR)_(50)-SK cells,and MitoSOX Red staining and PI staining were used to analyze mitochondrial ROS and apoptosis levels after phosphorylated AKT increased.Metformin was used to treat(GR)_(50)-SK and GFP CTRL-SK cells,respectively,and the apoptosis levels,mitochondrial ROS levels,mitochondrial morphology,AKT and its phosphorylation levels,and ATP concentrations of the two cells were detected by the above methods and ATP detection kit,respectively.Results·Western blotting showed that the construction of(GR)_(50)-SK cells was successful,and fluorescence microscopy showed that the construction of GFP CTRL-SK cells was also successful.PI staining results showed t
作者
冯奕源
徐忠匀
尹雅芙
王辉
程维维
FENG Yiyuan;XU Zhongyun;YIN Yafu;WANG Hui;CHENG Weiwei(Department of Nuclear Medicine,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China;Institute for Developmental and Regenerative Cardiovascular Medicine,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China)
出处
《上海交通大学学报(医学版)》
CAS
CSCD
北大核心
2023年第7期839-847,共9页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(81901162)
上海市青年科技启明星计划(20QA1406300)。