目的：建立HepG2（人肝胚胎瘤细胞）胰岛素抵抗（in-sulin resistance，IR）细胞模型，并在该模型上研究小檗碱改善IR 的效应。方法用25 mmol · L－1高糖，并分别用10－9、10－8、10－7、10－6、10－5、10－4 mol·L－1胰岛素处理，诱导HepG2细胞产生IR；对2-NBDG（荧光素标记葡萄糖）与HepG2细胞孵育浓度设定为：50、100、200、400、600、800μmol ·L－1、孵育时间设定为：20、40、60、80、100 min，拟筛选最佳胰岛素处理浓度、2-NBDG与HepG2最佳孵育浓度和最佳孵育时间，通过检测细胞培养液中葡萄糖的消耗量及细胞对葡萄糖的摄取量作为判定模型成功的标志；用二甲双胍、盐酸小檗碱干预模型细胞，研究盐酸小檗碱在细胞水平上改善IR的效应。结果6种浓度的胰岛素均可不同程度诱导HepG2产生IR，虽然10－5、10－4 mol·L－1剂量最明显，但细胞死亡较多，以10－6 mol·L－1剂量制模有效且细胞成活率高，与正常组比较差异具有统计学意义；当2-NBDG 与HepG2细胞孵育浓度大于100μmol·L－1时，细胞荧光强度与正常组差异有显著性，孵育时间超过20 min荧光强度与正常组比较有明显差异，超过100 min，细胞内荧光有明显淬灭衰减现象；盐酸小檗碱、二甲双胍能明显增加细胞培养上清液中葡萄糖的消耗及细胞对葡萄糖的摄取，与模型组比较差异具有统计学意义。结论用HepG2制作IR细胞模型时，选择10－6 mol·L－1剂量的胰岛素；2-NBDG孵育浓度选择为200μmol·L－1、2-NBDG孵育时间选择为80min较为适宜，盐酸小檗碱及二甲双胍对HepG2细胞IR具有明显的改善作用。%Aim To establish insulin resistance cell model on HepG2 cells (human embryonic liver tumor cells )and investigate the effect of berberine hydro-chloride on insulin-resistant HepG2 (IR-HepG2 ) cells.Methods ① IR model was induced by respec-tively using 10 -9 ,10 -8 ,10 -7 ,10 -6 ,10 -5 ,10 -4 mol ·L-1 insulin with 25 mmol · L-1 glucose in HepG2 cells.② HepG2 cells were incubated with 2-NBDG (fluorescent labeled glucose)in a series of concentra-tion:50,100,200,400,600,800 μmol·L-1 and a series of incubation time:20,40,60,80,100 min, to select the optimum concentration of insulin and the optimum incubation concentration and time of 2-NBDG in HepG2 cells.The success of the model was deter-mined by detecting the consumption of glucose in the cell supernatant and the uptake of glucose in HepG2 cells.③To study the effect of berberine hydrochloride on improving insulin resistance on the cell level,met-formin and berberine hydrochloride were used in the IR cells.Results Six concentrations of insulin induced the IR model in different degrees.Although 10 -4, 10 -5 mol·L-1 insulin was significant,a large amount of cells died.10 -6 mol·L-1 insulin was effective and had high survival rate of HepG2 cells,which had sta-tistical significance compared with the normal group. When the incubation concentration of 2-NBDG was more than 100 mol·L-1 ,the fluorescence intensity of the cells was significantly different from the normal group.When the incubation time of 2-NBDG was more than 20 min,fluorescence intensity was significantly different from the normal group.When the incubation time of 2-NBDG was more than 100 min,the fluores-cence quenching phenomenon was obvious in the cells. Berberine hydrochloride and metformin significantly in-creased the glucose consumption and glucose uptake in cell supernatant, which had statistical significance compared with the model group.Conclusions Using 10 -6 mol · L-1 insulin induced IR model in HepG2 cells,the optimum incubation concentration and incu-bation time of 2-NBDG is 200 μmol·L-1 and 80 min, respectively.Berberine hydrochloride and metformin have obvious effect on improving IR in HepG2 cells.
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