天津医药

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C2C12-GLUT4HA骨骼肌细胞株的筛选

仇利红1,高静2,牛文彦1   

  1. 1. 天津医科大学免疫教研室
    2. 天津中医药大学第一附属医院检验科
  • 收稿日期:2012-11-14 修回日期:2013-01-23 出版日期:2013-06-15 发布日期:2013-06-15
  • 通讯作者: 仇利红

Screening of C2C12-GLUT4HA Skeletal Muscle Cell Line

QIU Lihong 1,GAO Jing 2,NIU Wenyan 1   

  1. 1. Department of Immunology, Tianjin Medical University , Tianjin 300070, China
    2. Department of Clinical Laboratory,First Teaching Hospital of Tianjin University of Traditional Chinese Medicine
  • Received:2012-11-14 Revised:2013-01-23 Published:2013-06-15 Online:2013-06-15
  • Contact: QIU Lihong

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摘要： 【摘要】目的  筛选过表达带有HA表位的葡萄糖转运子4（GLUT4HA）且具有收缩能力的C2C12骨骼肌细胞株。方法  建立稳定过表达GLUT4HA的C2C12骨骼肌细胞株，从中挑选出23个克隆。用光镜观察各个克隆分化为多核肌管的能力和响应电脉冲刺激（EPS）的收缩能力，并用免疫印迹法检测各克隆中GLUT4HA蛋白的表达水平，筛选出分化良好、响应EPS收缩且高表达GLUT4HA的细胞株。将各细胞株进一步分为胰岛素组及其对照组、EPS组及其对照组，用酶联免疫吸附（ELISA）法测定各组细胞膜上GLUT4HA的含量（转位）。结果  在选出的23个克隆中，克隆17的GLUT4HA表达量较高，分化和收缩能力良好，且在胰岛素和EPS后GLUT4转位显著升高。结论  本研究建立了过表达GLUT4HA的具有收缩能力的C2C12-GLUT4HA骨骼肌细胞株，可用于研究胰岛素调节GLUT4转位和收缩调节GLUT4转位的机制。

关键词: 肌, 骨骼, 葡萄糖转运体4型, 细胞, 培养的, 胰岛素, 肌收缩, 电脉冲刺激

Abstract: Objective    To select a GLUT4HA-overexpressing C2C12 cell line with contractile activity. Methods    C2C12-GLUT4HA cell line stably overexpressing GLUT4HA was created and 23 of these clones were selected. The differentiation and contraction ability, as well as the expression level of GLUT4HA protein were detected.The clones with good myotubes ,contraction ability and high GLUT4HA protein level were divided into insulin group and its control, electrical pulse stimulation(EPS) group and its control,the amount of GLUT4HA on the cell surface was measured by enzyme-linked immunosorbent assay (ELISA) after stimulation by insulin and EPS. Results   Among the selected 23 clones, clone 17 has higher GLUT4HA protein level, good myotubes and contraction ability. In addition, GLUT4HA in clone 17 myotubes can translocate to cell surface under insulin and EPS stimulation. Conclusion     A contractile C2C12 muscle cell line stably overexpressing GLUT4HA was created. This cell line will be useful to study the mechanism of insulin- and contraction stimuli-regulated GLUT4 traffic.

Key words: muscle, skeletal, glucose transporter type 4, Insulin, muscle contraction, electrical pulse stimulation, 电脉冲刺激