TDP-43对氧糖剥夺诱导的小鼠HL-1心房肌细胞凋亡的影响及其机制  

Effect and mechanism of TDP-43 on oxygen-glucose deprivation-induced apoptosis in mouse HL-1 atrial myocytes cells

作　　者：岳娇 龚美婷 徐伍 王鹏 袁木 谭言飞[5] 裴海峰 Yue Jiao;Gong Mei-Ting;Xu Wu;Wang Peng;Yuan Mu;Tan Yan-Fei;Pei Hai-Feng(Clinical Medical College,Southwest Jiaotong University,Chengdu,Sichuan 610031,China;Department of Cardiology,4Health Service Training Center,Western Theater General Hospital of PLA,Chengdu,Sichuan 610083,China;Department of Urology,the Fifth Affiliated Hospital of Southern Medical University,Guangzhou,Guangdong 510900,China;National Engineering Research Center for Biomaterials,Sichuan University,Chengdu,Sichuan 610064,China;不详)

机构地区：[1]西南交通大学医学院,四川成都610031 [2]解放军西部战区总医院心内科,四川成都610083 [3]南方医科大学第五附属医院泌尿外科,广东广州510900 [4]解放军西部战区总医院卫勤训练中心,四川成都610083 [5]四川大学国家生物医学工程研究中心,四川成都610064

出　　处：《解放军医学杂志》2023年第11期1305-1313,共9页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81970241);西部战区总医院院管重点项目(2021-XZYG-A03)。

摘　　要：目的探讨TARDNA结合蛋白43(TDP-43)对氧糖剥夺(OGD)诱导小鼠HL-1心房肌细胞凋亡的影响及其机制。方法将体外培养的小鼠HL-1心房肌细胞分为:(1)对照组及不同OGD处理时间(2、4、8、16h)组,采用CCK-8法检测细胞活力,Westernblotting检测TDP-43蛋白表达水平,以此确定OGD诱导时间点用于后续研究;(2)对照组与OGD组,采用流式细胞术检测细胞凋亡率,JC-1染色法检测线粒体膜电位,化学发光法检测三磷酸腺苷(ATP)相对含量,微板法检测丙二醛(MDA)含量,WST-1法检测超氧化物歧化酶(SOD)含量。将转染慢病毒的小鼠HL-1心房肌细胞分为:(1)阴性对照慢病毒干预组(NC-shRNA)、TDP-43敲低慢病毒干预组(TDP-43-shRNA1、TDP-43-shRNA2、TDP-43-shRNA3),Westernblotting检测TDP-43蛋白表达水平,选择慢病毒敲低效率最高的TDP-43-shRNA用于后续试验;(2)NC-shRNA组、TDP-43-shRNA组、OGD+NC-shRNA组、OGD+TDP-43-shRNA组,在常氧条件和OGD条件下,采用流式细胞术检测细胞凋亡率,MitoTracker染色法检测线粒体形态,JC-1染色法检测线粒体膜电位,化学发光法检测ATP含量,流式细胞术检测活性氧(ROS)荧光强度,微板法检测MDA含量,WST-1法检测SOD含量。结果CCK-8法检测结果显示,随OGD时间的延长,小鼠HL-1心房肌细胞的活力逐渐下降;Westernblotting检测结果显示,TDP-43蛋白表达水平逐渐升高,两者均呈现较强的时间依赖性。与对照组比较,在OGD16h时小鼠HL-1心房肌细胞活力最低(P<0.05)、TDP-43蛋白表达量最高(P<0.05),据此后续实验选择OGD16h为诱导时间点。与对照组比较,OGD组细胞凋亡率、线粒体膜电位荧光强度比值、MDA含量升高,ATP相对含量、SOD含量降低,差异均有统计学意义(P<0.05)。Westernblotting检测结果显示,与NC-shRNA组比较,TDP-43-shRNA2组TDP-43蛋白表达水平降低最为明显(P<0.05),敲低效率最高,因此选择TDP-43-shRNA2进行后续实验。流式细胞术检测结果显示,在常氧条件下,与NC-Objective To investigate the effects and mechanisms of TAR DNA-binding protein 43(TDP-43)on oxygen-glucose deprivation(OGD)-induced apoptosis in mouse atrial myocytes(HL-1 cells).Methods The in vitro cultured mouse atrial myocytes(HL-1 cells)were divided into:(1)control group and groups with different OGD treatment times(2,4,8,16 h),and cell viability was detected by CCK-8 assay,and TDP-43 protein expression level was detected by Western blotting,which was used to determine the time point of OGD induction for the subsequent study;(2)control and OGD groups,flow cytometry was used to detect apoptosis,JC-1 staining to detect mitochondrial membrane potential,chemiluminescence to detect adenosine triphosphate(ATP)relative content,microplate method to detect malondialdehyde(MDA)content,and WST-1 method to detect superoxide dismutase(SOD)content.Mouse atrial myocytes(HL-1 cells)transfected with lentivirus were divided into:(1)negative control lentiviral intervention group(NC-shRNA),TDP-43 knockdown lentiviral intervention group(TDP-43-shRNA1,TDP-43-shRNA2,TDP-43-shRNA3),and Western blotting was used to detect the TDP-43 protein expression level,and the group with the highest lentiviral knockdown efficiency was selected as the TDP-43-shRNA for subsequent experiments;(2)NC-shRNA group,TDP-43-shRNA group,OGD+NC-shRNA group,OGD+TDP-43-shRNA group,under normoxic and OGD conditions,flow cytometry was used to detect the apoptosis rate,MitoTracker staining to detect mitochondrial morphology,JC-1 staining to detect mitochondrial membrane potential,chemiluminescence to detect the relative content of ATP,flow cytometry to detect the fluorescence intensity of reactive oxygen species(ROS),microplate to detect the content of MDA,and WST-1 to detect the content of SOD.Results CCK-8 method showed that,with the prolongation of OGD time,the viability of mouse atrial myocytes(HL-1 cells)gradually decreased;Western blotting assay showed that the expression level of TDP-43 protein gradually increased,and both of them showed a strong time-de

关 键 词：TARDNA结合蛋白43 氧糖剥夺 细胞凋亡 氧化应激 

分 类 号：R542.2[医药卫生—心血管疾病]