中国实验动物学报2025，Vol.33Issue(5)：684-694,11.DOI:10.3969/j.issn.1005-4847.2025.05.007
急性高原低氧诱导心肌损伤小鼠模型免疫印迹实验内参蛋白的选择
Loading control for Western Blot in myocardial injury models induced by acute high altitude
摘要
Abstract
Objective The myocardial injury was induced by hypobaric hypoxia through regulating the expression of various proteins.The expression of proteins was mainly detected by western blot,but the selection of internal reference proteins and their variations have not been systematically studied.Methods Myocardial injury was induced in a low-pressure,low-oxygen chamber simulating an altitude of 6000 m,for 24 and 72 h.Establishment of the myocardial injury model was confirmed by hematoxylin eosin(HE)staining.Expression levels of internal control proteins,including vinculin,α-tubulin,eukaryotic translation initiation factor-5(EIF5),β-actin,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),cyclophilin B,and cofilin,were detected by Western Blot and total protein expression was detected by Ponceau S and Coomassie Blue staining.An adult mouse cardiomyocytes(AMCMs)injury model was induced by hypoxia for 12 and 24 h and confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL staining).Internal control proteins were detected by Western Blot,as in the in vivo model,and total protein expression was detected by Ponceau S and Coomassie Blue staining.Results A myocardial injury model was established by hypobaric hypoxia for 24 and 72 h,the total protein expression levels remained consistent.The expression of internal control proteins including vinculin,EIF5,β-actin,cyclophilin B,and cofilin was consistent between the control and model groups.Expression levels of α-tubulin were similar in the plain control and 24 h hypobaric hypoxia group,but were significantly lower in the 72 h hypobaric hypoxia group compared with the plain control group.GAPDH expression was significantly higher in the 24 and 72 h hypobaric hypoxia groups than in the plain control group.An AMCM injury model was established by hypoxia for 12 and 24 h.Total protein levels and expression levels of the internal control proteins EIF5 and β-actin were consistent,but vinculin,α-tubulin,GAPDH,cyclophilin B,and cofilin expression levels were higher in both hypoxia groups compared with the normoxic control group.Conclusions EIF5 and β-actin may be the suitable loading control proteins for studies of hypobaric hypoxia-induced myocardial injury using Western Blot.Total protein is also a good choice for hypobaric hypoxia studies.关键词
高原低氧/心肌细胞损伤/免疫印迹实验/内参蛋白Key words
hypobaric hypoxia/myocardial injury/Western Blot/loading control proteins分类
生物学引用本文复制引用
张小玉,张依曼,刘佳,来宝长,乌仁塔娜,田红燕,殷倩..急性高原低氧诱导心肌损伤小鼠模型免疫印迹实验内参蛋白的选择[J].中国实验动物学报,2025,33(5):684-694,11.基金项目
西安交通大学第一附属医院院基金(2023GYZX02,2018QN-04),西安交通大学基本科研业务费(xzy012023131).Funded by hospital foundation of the Frist Affiliated Hospital of Xi'an Jiaotong University(2023GYZX02,2018QN-04),Basic Scientific Research Fund of Xi'an Jiaotong University(xzy012023131). （2023GYZX02,2018QN-04）
