基于柱前衍生化高效液相色谱法评价骨骼肌细胞胰岛素抵抗模型

Evaluation of insulin resistance model in skeletal muscle cells based on high performance liquid chromatography with precolumnderivatization

培养C2C12成肌细胞并分化成骨骼肌细胞,取细胞培养液通过柱前衍生化,将葡萄糖与1-苯基-3-甲基-5-吡唑啉酮(PMP)络合,使用Unisphere Aqua C18色谱柱(2.0mm×150mm,粒径3.5μm)分离衍生化产物,流动相为V(10mmol/L乙酸铵(pH=5.30))∶V(乙腈溶液)=78∶22,流速为1.0mL/min,检测波长为245nm,进样量为20μL,柱温30℃.葡萄糖质量浓度在100~500mg/L范围内具有良好的线性,相关系数0.998 5;精密度、重复性良好,平均回收率100.26%,RSD值为1.02%.地塞米松孵育处理的骨骼肌细胞在胰岛素刺激下对葡萄糖的摄取量与对照组相比显著减少.结果显示:柱前衍生化高效液相色谱法可用于评价骨骼肌细胞胰岛素抵抗模型,为后续胰岛素抵抗机制探讨及药物筛选打下基础.

C2C12 cells were cultured in DMEM and differentiated in different ionmedium. The glucose in the culture medium was derivatized with 1-phenyl-3-methyl-5-pyrazolone （PMP） and the resulting samples were analyzed by HPLC using an Unisphere Aqua C18 column. The mobile phase was V（10 mmol/L ammonium acetate buffer （pH = 5. 30）） ： V （ acetonitrile） = 78 ： 22.The flow rate was 1.0 mL/min and the detection wavelength was 245 nm. The column temperature was 30 C and injection volume was 20 μL. The glucose derivative was well separated,and presented a good linear relation （r2 =0. 998 5） in the concentration range of 100＂500 mg/L. Average recovery rate was 100. 26%, and RSD was 1.02%. After treatment with dexamethasone,the amount of glucose uptake was significantly reduced under insulin stimulation compared with control group. In conclusion,HPLC with precolumn derivatization can be utilized for assessing insulin resistance model in skeletal muscle cells, which provides basis forinvestigation of mechanisms of insulin resistance and drug screening.