大鼠骨骼肌中19S调节复合体的分离、提纯及其在严重烫伤后蛋白水平的变化

The isolation and purification of 19S regulator compound and the change of its protein level in rat skeletal muscle after severe scalding

目的 探讨 19S调节复合体蛋白在烫伤大鼠骨骼肌蛋白降解中的作用机制。 方法 利用层析技术 ,分离、纯化大鼠骨骼肌 19S调节复合体 ,并常规免疫制备其抗体。制作Wistar大鼠烫伤模型 ,随机分为烫伤组 ,设伤后 1、2、3、5、7d时相点 (每时相点 8只 ) ,另取 8只正常大鼠设为对照组。用固相免疫印迹法测定 19S调节复合体含量的变化。另取 6 4只烫伤大鼠 ,行 19S抗体注射实验 ,分为 :注射组 ,由尾静脉分两次注射纯化的 19S抗体 ( 3mg/kg) ,分别于注射后第 1、2、3天处死 ;对照组 ;尾静脉注射相同体积等渗盐水 ,其余处理同注射组。测定烫伤大鼠骨骼肌中酪氨酸的释放速率。 结果 获得了纯度较高的 19S调节复合体。从烫伤后第 1天起 ,与对照组比较 ,大鼠骨骼肌中的 19S调节复合体含量开始明显增加 ,骨骼肌蛋白质降解速率也在伤后第 2天显著增强 ,19S抗体则能明显抑制蛋白质降解的增加。 结论 烧伤刺激可上调骨骼肌 19S调节复合体的蛋白水平 ,从而激活 2 6S蛋白酶复合体对蛋白质的降解作用 ,这可能是烧伤后负氮平衡的发生重要原因。

Objective To explore the role of 19S regulator compound protein in the degradation of skeletal muscle protein in scalded rats. Methods Wistar rats were scalded and they were randomly divided into normal and 1, 2, 3, 5, and 7 postburn day (PBD) groups with 8 rats in each group. The 19S regulator compound of skeletal muscle in scalded rats was isolated and purified with chromatography. Rabbit-anti-rat antibody IgG of 19S regulator compound was prepared conventionally. The antibody was injected to rats in injection group (I) in which 19S antibody in dose of 3 mg/kgBW was injected for two times via tail vein with 6-hour interval. The rats in I group were decapitated on 1,2 and 3 post-injection days, respectively. The scalded rats in control group (C) were treated in the same way, except that the antibody was replaced by normal saline. The change in content of 19S regulator compound was determined by western-blot. Meanwhile, the releasing rate of tyrosine from the skeletal muscle of scalded rats was also detected by fluorescent photography. Results 19S regulator compound with high purity was obtained. The content of 19S regulator compound in rat skeletal muscle was increased significantly after 2 PBD. But the protein degradation rate was also obviously increased on 2 PBD. The antibody of 19S compound might inhibit the enhancement of protein degradation. Conclusion Burn injury might up-regulate the protein level of skeletal muscle 19S regulator compound, which therefore activated the protein degradation by 26S protease compound. This might be an important factor leading to postburn negative nitrogen balance. [