Mst-1调控缺氧复氧诱导的小鼠心肌细胞自噬及凋亡的机制  

Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse

作　　者：王艳 赵然尊 仇治梅 沈长银 陈攀科 郝星 袁近松 邓文文 石蓓 Wang Yan;Zhao Ranzun;Qiu Zhimei;Shen Changyin;Chen Panke;Hao Xing;Yuan Jinsong;Deng Wenwen;Shi Bei(Department of Cardiology,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,China;Department of Cardiology,Second Affiliated Hospital,Zunyi Medical University,Zunyi 563000,China)

机构地区：[1]遵义医科大学附属医院心血管内科,563000 [2]遵义医科大学第二附属医院心血管内科,563000

出　　处：《中华心血管病杂志》2020年第12期1060-1069,共10页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81860061);贵州省科学技术基金[黔科合LH字(2014)7576号];遵义市科技支撑项目[遵义合字(2016)06号];遵义医科大学附属医院硕士科研启动基金[院字(2016)51号]。

摘　　要：目的探索小鼠心肌细胞中哺乳动物不育系20样激酶1(mammalian sterile 20-like kinase 1,Mst-1)调控缺氧复氧(hypoxia reoxygenation,HR)诱导的心肌细胞自噬及凋亡机制。方法采用酶消化法结合差速贴壁的方法培养小鼠乳鼠心肌细胞,通过缺氧24 h复氧6 h的方法建立HR模型。实验分组:对照组:正常培养的心肌细胞;Mst-1空病毒组:用重组慢病毒空载体转染心肌细胞48 h;Mst-1敲低组:用携带Mst-1小干扰RNA(siRNA)的重组慢病毒转染心肌细胞48 h;Mst-1过表达组:用携带Mst-1基因的重组慢病毒转染心肌细胞48 h;HR组:建立心肌细胞HR模型;Mst-1敲低+HR组:用携带Mst-1 siRNA的重组慢病毒转染心肌细胞后48 h建立心肌细胞HR模型;Mst-1过表达+HR组:用携带Mst-1基因的重组慢病毒转染心肌细胞后48 h建立心肌细胞HR模型。采用实时荧光定量RCR(qPCR)以及Western blot检测细胞中Mst-1 mRNA及蛋白相对表达量,免疫荧光染色检测心肌细胞标志物心肌肌钙蛋白T(cTnT),及细胞自噬体与自噬溶酶体变化,脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法检测心肌细胞凋亡水平,Western blot检测自噬相关蛋白微管相关蛋白1轻链3(LC3)Ⅱ/LC3Ⅰ,P62及凋亡相关蛋白半胱氨酸天冬氨酸酶剪切体(cleaved-caspase)9、半胱氨酸天冬氨酸酶前体(pro-caspase)9、cleaved-caspase-3、pro-caspase-3,以及髓细胞白血病1基因(MCL-1)的表达情况达。予以MCL-1抑制剂A1210477做回复实验,验证Mst-1通过调控MCL-1参与心肌细胞凋亡及自噬。结果免疫荧光检测发现培养细胞表达心肌细胞特异性标志物cTnT;HR情况下心肌细胞中Mst-1表达增加,慢病毒转染可有效抑制或过表达细胞中Mst-1。HR组与Mst-1过表达+HR组心肌细胞内自噬体及自噬溶酶体含量低于对照组,而Mst-1敲低+HR组心肌细胞内自噬体及自噬溶酶体含量高于HR组(P均<0.05)。TUNEL结果显示,HR组及Mst-1过表达+HR组中TUNEL阳性细胞比例高�Objective To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation(HR)induced myocardial cell autophagy and apoptosis.Methods Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells.HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours.The experimental groups including control group(normal cultured cardiomyocytes),Mst-1 empty virus group(cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours),Mst-1 knockdown group(recombinant lentivirus carrying Mst-1small interfering RNA(siRNA)was transfected into cardiomyocytes for 48 hours),Mst-1 overexpression group(cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours),HR group(cardiomyocytes exposed to HR),Mst-1 knockdown+HR group(HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA)and Mst-1 overexpression+HR group(HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene).Real-time fluorescence quantitative RCR(qPCR)and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells,immunofluorescence staining was used to detect cardiomyocyte troponin T(cTnT),and autophagosomes and autophagy enzyme changes.TUNEL method was used to detect myocardial cell apoptosis,Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3(LC3)Ⅱ/LC3Ⅰ,P62 and apoptosis-related protein cleaved-caspase 9,pro-caspase 9,cleaved-caspase-3,pro-caspase-3,and myeloid leukemia 1(MCL-1)expression.MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy.Results Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT.The expression of Mst-1 in cardiomyocytes increas

关 键 词：肌细胞 心脏 哺乳动物不育系20样激酶1 自噬 凋亡 髓细胞生长因子1 

分 类 号：R54[医药卫生—心血管疾病]