大鼠骨髓间充质干细胞条件培养基对HepG2细胞胰岛素抵抗的影响

Effects of conditioned media for rat bone marrow-derived mesenchymal stem cells on palmitic acid-induced insulin resistance in HepG2 cells

目的 观察大鼠骨髓间充质干细胞条件培养基（BMSCs-CM）对人HepG2细胞胰岛素抵抗（IR）的影响,并探讨其分子机制.方法 根据葡萄糖利用情况筛选出最佳造模剂使用浓度及时间;以正常HepG2细胞为空白对照,给予棕榈酸处理的细胞为IR模型组,加入BMSCs-CM为条件培养基（CM）干预组,而加入同样经半透膜浓缩后的低糖培养基作为阴性对照组,分别采用细胞培养、糖原染色、葡萄糖及糖原检测试剂盒等方法来分析各组葡萄糖利用情况;免疫印迹法观察BMSCs-CM对IR HepG2细胞磷酸化的胰岛素受体底物（p-IRS）、磷脂酰肌醇-3-激酶（PI3K）与磷酸化的丝氨酸/苏氨酸蛋白激酶（p-AKT）蛋白表达的影响;分别加入PI3K抑制剂LY294002及激动剂740Y-P后继续观察上述胰岛素信号蛋白的变化情况.结果 （1）以0.25 mmol/L棕榈酸作用HepG2细胞24 h的方法成功建立IR模型;IR模型组培养液中葡萄糖含量明显高于空白对照组,其细胞内糖原含量也显著减少（P＜0.05）.（2）与IR模型组相比,CM干预组HepG2/IR葡萄糖的摄取与利用明显改善（P＜0.05）,而在阴性对照组则差异没有统计学意义.（3）CM干预组p-IRS、PI3K与p-AKT的蛋白表达水平显著高于IR模型组（P＜0.05）;而在加入LY294002阻断PI3K后,给予BMSCs-CM后PI3K及p-AKT蛋白含量则未增加;进一步加入740Y-P过度激活PI3K后,则这些蛋白的表达水平较IR模型组显著升高.结论 大鼠BMSCs-CM的胰岛素增敏作用与p-IRS、PI3K与p-AKT蛋白的表达增强有关.

Objective To study the effect of conditioned media for rat bone marrow mesenchymal stem cells （BMSCs-CM） on palmitic acid （PA）-induced insulin resistance （IR） in HepG2 cells and its underlying molecular mechanisms.Methods HepG2 cells were treated with or without BMSCs-CM and L-DMEM in the presence or absence of PA.Glucose utilization in HepG2 cells were detected with PAS,glucose and glycogen measurements.Western blotting was used to assess the expression of phospho-insulin receptor substrate （p-IRS）,phosphatidylinositol 3-kinase （PI3 K） and p-AKT.Results （1） Incubation of HepG2 cells with 0.25 mmol/L PA for 24 hours significantly increased the glucose concentration and decreased the glycogen content （P 〈 0.05） in the media.（2） Treatment with BMSCs-CM significantly ameliorated the glucose and glycogen alteration in cells pretreated with PA （P 〈 0.05）,however,no obvious effect of BMSCs-CM on the cell glucose and glycogen production.（3） BMSCs-CM treatment also increased protein expression of p-IRS,PI3K and p-AKT in PA incubated HapG2 cells （P〈 0.05）.The effect of BMSCs-CM on PI3K and p-AKT expression could be mimicked upon addition of 740Y-P,a PI3K agonist,but abolished by LY294002,a PI3K specific inhibitor.Conclusions BMSCs-CM could improve the insulin sensitivity in HepG2 cells pretreated with PA through upregulation of insulin signaling component expression.