双向电泳分析失神经支配大鼠腓肠肌蛋白的表达变化

Protein Expression of Denervated Rat Gastrocnemius by Two-dimensional Gel Electrophoresis

目的:建立骨骼肌蛋白分离的双向电泳(2-DE)技术体系,寻找失神经支配骨骼肌与正常骨骼肌蛋白的表达差异。方法:建立失神经腓肠肌模型,提取腓肠肌总蛋白,进行第一向等电聚焦(IEF)和第二向十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分离总蛋白,使用PDQuest软件对凝胶图像进行分析,并测量了凝胶蛋白斑点在IEF和SDS-PAGE方向上的位置偏差。结果:失神经组和正常组腓肠肌组织3块凝胶的平均蛋白质点数分别为560±16和545±13,平均匹配点数分别为471±19和447±17,匹配率达84.1%和82.1%。对比分析了两种腓肠肌组织的双向电泳图谱发现,平均匹配点数为445±8;发现在失神经支配后有80个点发生了明显而稳定的质和量(大于10倍或小于0.1倍)的改变。不同凝胶间蛋白质点在IEF方向的偏差为(1.203±0.763)mm,在SDS-PAGE方向上的偏差为(1.062±0.529)mm。结论:失神经组和正常组腓肠肌的双向电泳图谱存在明显差异,失神经后表达下调的蛋白可能与维持肌肉正常功能相关,而表达上调的蛋白则可能与肌萎缩的发生过程相关。

Objective:To establish two-dimensional gel electrophoresis(2-DE) system for skeletal muscle, and find differences of gastrocnemius protein of denervated rat and control group.Methods:Establish the model of denervated gastrocnemius, extract the total proteins from the denervated gastrocnemius tissue and normal gastrocnemius tissue, and run isoelectric focusing(IEF) electrophores as the first dimension , the sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE) as the second dimension to separate the total proteins . Protein spots were visualized by Bio-safe Coomassie and the image were abtained through the GS-800 scanning. The protein spots were analyzed by PDQuest software and measure the position deviation of matched spots in IEF and SDS-PAGE.Results:For normal gastrocnemius tissue, the average protein spots of 3 gels were 560±16; and 471±19 spots were matched with the average matching rate of 84.1%. For denervated gastrocnemius tissue, the average protein spots of 3 gels were 545±13; and 447±17 spots were matched with the average matching rate of 82.1%.A total of 445±8 spots were matched between the electrophoretic maps of 3 denervated gastrocnemius tissue and paired normal gastrocnemius tissue. A good reproducibility of spot position 2-DE map was obtained. 80 spots were found significantly changed in quality and quantity in the group of denervation. The average position deviation of matched spots was 1.203±0.763 mm in IEF direction, and 1.062±0.529mm in SDS-PAGE direction.Conclusions:There are distinctive differences between 2-DE maps of denervated gastrocnemius and control group,downregulated proteins may be involved in keeping the normal function of muscle and upregulated proteins may participate in the process of atrophy after denervation.