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骨骼肌细胞葡萄糖运载体4的实验研究 |
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杨晓冰1 吴 毅1 李云霞1 胡永善1 张新堂 2 平蓓芳2 朱尚权2
摘要 目的：制备大鼠骨骼肌葡萄糖运载体4(glucose transporter 4,GLUT4)羧基端13肽的多克隆抗体，分离正常大鼠骨骼肌细胞内、外膜，并测定膜上GLUT4的含量。方法：采用Merrifield固相合成法制备GLUT4羧基端13肽，免疫兔子后得到抗血清和抗体。运用蔗糖梯度超速离心法分离大鼠骨骼肌细胞内外膜，并以Western印迹法测定细胞内外膜GLUT4蛋白的含量。结果：正常大鼠骨骼肌外膜和内膜中均有GLUT4的存在，其中内膜中的含量大于外膜。结论：运用免疫法获得的GLUT4多克隆抗体具有高效价，对骨骼肌细胞内外膜GLUT4的研究，将有助于糖代谢紊乱、葡萄糖转运机制障碍的研究。
关键词 多克隆抗体； 骨骼肌； GLUT4； Western印迹法
An experimental study of the distribution of GLUT4 in rat skeletal muscle/YANG Xiaobing , WU Yi, LI Yunxia, et al//Chinese Journal of Rehabilitation Medicine,1998,13(3):98～100
Abstract Objective:To prepare the polyclonal antibodies against the peptide corresponding carboxyl terminus of GLUT4,and study the distrubution of GLUT4 glucose carriers in normal SD rat′s skeletal muscle. Method:The 13-aa peptide corresponding carboxyl terminus of GLUT4 was prepared by Merrifield′s solid phase synthesis.After immuning rabbits with immunogen which was mixed 13-aa peptide with typhoid bacillus, we tested polyclonal antibody with ELISA. The distribution of GLUT4 glucose carriers in skeletal muscle which was seperated by using sucrose gradient ultracentrifugation was examined with Western-blot analysis. Result:The polyclonal antibody had high titer. There were more GLUT4 in intracellular membrane fraction than membrane preparation enriched in sarcolemma.Conclusion:Polyclonal antibody against the peptide corresponding carboxyl terminus of GLUT4 we prepared mihgt provide quantified assessment of curative effect for diabetes.
Author′s address Huashan Hospital, Shanghai Medical University, Shanghai, 200040
Key words Polyclonal antibody;Skeletal muscle;Glucose transporter 4; Western-blot
研究表明，葡萄糖的跨膜转运是骨骼肌葡萄糖代谢的限速步骤，这一过程是由细胞膜上的葡萄糖运载体（glucose transporter, GLUT)介导并通过易化扩散来完成的〔1〕。目前发现骨骼肌内存在两种GLUT，分别为GLUT1和GLUT4。前者活性低，只在基础状态下起作用；而后者活性高，在基础状态及胰岛素刺激下都起主要的转运葡萄糖的作用。通常GLUT4存在于细胞内膜中。在胰岛素的作用下，GLUT4从细胞内膜转位至细胞外膜，从而发挥其生理作用〔2〕。骨骼肌是全[1] [2] [3] 下一页
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