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
李笑汝 1姚新叶 2刘佳 1张小玉 3张依曼 4来宝长 1马强 5王一东 1田红燕 5殷倩6
作者信息
- 1. 西安交通大学心血管研究中心,西安 710061
- 2. 西安交通大学心血管研究中心,西安 710061;西安交通大学第一附属医院心血管内科,西安 710061
- 3. 西安交通大学第一附属医院周围血管科,西安 710061
- 4. 西安交通大学第一附属医院周围血管科,西安 710061;西安交通大学心血管研究中心,西安 710061
- 5. 西安交通大学第一附属医院周围血管科,西安 710061;西安交通大学第一附属医院心血管内科,西安 710061
- 6. 西安交通大学第一附属医院周围血管科,西安 710061;西安交通大学心血管研究中心,西安 710061;西安交通大学第一附属医院心血管内科,西安 710061
- 折叠
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 72h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 2h相比,贴壁后 24 和 48h的细胞存活率无显著下降,贴壁后 72h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 48h 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.
Abstract
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 for α-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Key words
AMCMs extraction/adenovirus transfection/hypoxia-reoxygenation引用本文复制引用
基金项目
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项目(2018QN-04)
出版年
2024
国家科技期刊平台
NSTL
万方数据