成年小鼠心肌细胞腺病毒转染及缺氧/复氧诱导损伤模型的建立  

Models of adenoviral transfection and hypoxia/reoxygenation-induced injury in AMCMs of adult mouse cardiomyocytes

作　　者：李笑汝 姚新叶 刘佳[2] 张小玉 张依曼 来宝长[2] 马强[1,3] 王一东 田红燕[1,3] 殷倩 LI Xiaoru;YAO Xinye;LIU Jia;ZHANG Xiaoyu;ZHANG Yiman;LAI Baochang;MA Qiang;WANG Yidong;TIAN Hongyan;YIN Qian(Department of Peripheral Vascular,the First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China;Cardiovascular Research Center,Xi’an Jiaotong University,Xi’an 710061,China;Department of Cardiovascular Medicine,the First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China)

机构地区：[1]西安交通大学第一附属医院周围血管科,西安710061 [2]西安交通大学心血管研究中心,西安710061 [3]西安交通大学第一附属医院心血管内科,西安710061

出　　处：《中国实验动物学报》2024年第4期435-443,共9页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金青年基金(81900256);西安交通大学第一附属医院院基金(2018QN-04)。

摘　　要：目的 采用非Langendorff方法分离成年小鼠心肌细胞(adult mouse cardiomyocytes, AMCMs),并建立腺病毒转染及缺氧/复氧诱导细胞损伤模型,为应用AMCMs进行心肌缺血体外实验的研究提供便捷、实用、可操作性强的系统方法。方法 应用非Langendorff方法分离AMCMs,观察贴壁后2、24、48及72 h细胞的形态,计算存活率,并应用α-actinin免疫荧光染色观察AMCMs完整性;应用腺病毒转染AMCMs,观察并统计转染36、48 h后的转染效率;通过缺氧45 min/复氧24 h,进行碘化丙啶(propidium iodide, PI)染色,统计PI阳性细胞率以明确缺氧/复氧损伤模型的建立。结果 与AMCMs贴壁后2 h相比,贴壁后24和48 h的细胞存活率无显著下降,贴壁后72 h的细胞存活率显著下降;AMCMs细胞结构完整,可进行腺病毒转染,36和48 h转染效率分别可达82.07%±0.60%和82.84%±1.18%;缺氧/复氧诱导AMCMs可成功建立细胞损伤模型,PI染色阳性细胞达42.28%±3.10%。结论 本研究应用非Langendorff方法分离培养的AMCMs,细胞存活率高,并建立腺病毒转染和缺氧/复氧诱导细胞损伤模型,为体外建立AMCMs基因修饰、缺氧/复氧损伤模型提供了简单易行的系统方法。Objective To construct models of viral transfection and hypoxia/reoxygenation induced cellular injury in adult mouse cardiomyocytes(AMCMs)isolated using a non-Langendorff method.Methods AMCMs were isolated,extracted,sedimented,and plated using a non-Langendorff method.The morphology and survival rate of the isolated cells were evaluated 2,24,48 and 72 h after plating,and their integrity was observed by immunofluorescence staining forα-actinin.The isolated AMCMs were infected with adenoviruses carrying an RFP-expressing vector and fluorescence images were obtained at 36 and 48 h post-infection and used to calculate transfection efficiency.The cells were cultured under hypoxic conditions for 45 min,reoxygenated for 24 h,and then stained with propidium iodide(PI)to verify establishment of the hypoxia/reoxygenation injury model.Results The survival rates of AMCMs at 2,24 and 48 h after plating were comparable,but survival was significantly reduced at 72 h.The integrity of the AMCMs was good and>80%of the cells were transfected with adenovirus at 48 h.After hypoxia/reoxygenation treatment,42%of cells were stained by PI,suggesting successful establishment of the AMCM injury model.Conclusions In this study,we developed a non-Langendorff method for the fast and easy isolation of AMCMs with high cell viability.The isolated cells can be efficiently infected with adenovirus and respond to hypoxia/reoxygenation injury.These findings provide a systematic method for isolating AMCMs and for applying gene modification and hypoxia/reoxygenation injury in these cells.

关 键 词：AMCMs提取 腺病毒转染 缺氧/复氧 

分 类 号：Q95-33[生物学—动物学]