优化心肌膜蛋白提取方法及心肌缺血时间隙连接蛋白43的表达变化(英文)

Optimized method for extracting membrane proteins from the myocardium and its application in the detection of connexin 43 expression changes under myocardial ischemia

目的优化膜蛋白提取方法,以便研究心肌缺血时connexin43(间隙连接蛋白43)的表达变化。方法①差速离心法结合溶剂提取法提取心肌膜蛋白,溶解膜蛋白时比较了6组方案,通过各组方案在聚丙烯酰胺凝胶电泳中条带分布差异筛选出最佳方案,分析心肌细胞膜表面受体表达。②通过左冠脉结扎造成对照组、5min、20min、60min心肌缺血组,研究缺血时心肌connexin43表达变化。结果①最终选择RIPA含4%SDS来溶解膜蛋白,并成功检测出connexin43,βactin(管家蛋白),M受体(毒蕈碱型乙酰碱受体)等膜蛋白。②免疫印迹技术检测缺血不同时相connexin43表达变化,5min、20min、60min缺血组相对对照组分别减少20%、60%、76%。结论应用此方案能成功提取心肌膜蛋白,为进一步研究心肌缺血时connexin43的表达变化奠定了基础。

Objective To establish optimized method for extracting membrane proteins from the myocardium which will facilitate the study of connexin 43 expression under myocardial ischemia. Methods ①The method that combined differential centrifuging and solvent adding was applied in extracting membrane proteins from the myocardium. Six strategies for solubilizing membrane proteins were compared through PAGE （polyacrylamide gel electropheresis） to establish optimized method. The expression of membrane proteins was detected. ②By the treatment of left coronary artery occlusion, acute myocardial ischemia occurred in rats in four groups： control, 5 min ischemia, 20min ischemia, and 60min ischemia. The expression of connexin 43 according to ischemia time course. Results ①We found that radioimmunoprecipitation （RIPA） buffer containing 4% SDS （sodium dodecyl sulphate） was the optimized recipe to solubilize membrane proteins. Through immunoblotting, counexin 43, β-actin, and M receptors （muscarinic acetylcholine receptors） were successfully detected. ②The quantity of connexin 43 in the four groups decreased respectively following the ischemic time course. The percentage of decreasing in each ischemia group compared to that of the control group was 20 %, 60 % and 76 %, respectively. Conclusion We have established the optimized method for extracting membrane proteins from the myocardium which will contribute to the study of counexin 43 expression under myocardial ischemia.