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目的研究不同浓度的葡萄糖和游离脂肪酸(FFA)对骨骼肌L6细胞胰岛素敏感性及细胞内活性氧(ROS)的影响。方法 L6细胞诱导分化成熟后,分为6组,以5 mmol/L葡萄糖为低糖对照组,其中加0.3或1 mmol/L的混合FFA为低糖低FFA或低糖高FFA组,以25 mmol/L葡萄糖为高糖对照组,其中加0.3和1 mmol/L的FFA的培养基为高糖低FFA或高糖高FFA组。干预48 h,~3H-D-葡萄糖摄取实验(加与不加100 nmol/L胰岛素37℃30 min)验证L6细胞胰岛素敏感性,经荧光探针H2DCFDA观察细胞内ROS含量的变化。结果在5 mmol/L葡萄糖组,低糖对照组基础摄糖值为22777.6±6608.7,低糖低FFA组为26027.5±4085.7,低糖高FFA组为146805.1±21099.4。胰岛素刺激后,低糖对照组为76586.5±18450.1,低糖低FFA组为43738±9203.6,低糖高FFA组为32050.6±3362.3,较低糖对照组显著降低(P<0.01)。在25 mmol/L葡萄糖组,基础葡萄糖摄取量为11793.8±551. 4,高糖低FFA组11887.3±2082.3高糖高FFA组为13886.1±3872.9,差别无统计学意义(P>0.05),但较低糖对照组显著降低(P<0.01);加入胰岛素刺激后,高糖对照组为42798.8±9441.5,高糖低FFA组为23946.9±3839.6,高糖高FFA组为13886.1±3872.9,各组之间差别显著(P<0.01)。对细胞内ROS的研究发现,低糖低FFA组为2234.2±477.2,低糖高FFA组为1969.0±480.9,高糖对照组为1969.0±229.4,高糖低FFA组为2184.5±734.2,高糖高FFA组为2571.3±96.7,可以显著增加的L6细胞内ROS,与低糖对照组(615.3±244.3)相比差别显著(P<0.01)。除对照组之外的各组之间差别无统计学意义(P>0.05)。结论高糖和高FFA可以诱导L6细胞出现氧化应激,同时高糖和高FFA是导致骨骼肌细胞胰岛素抵抗的重要原因之一。
Objective To investigate the effects of different concentrations of glucose and free fatty acid (FFA) on insulin sensitivity and intracellular reactive oxygen species (ROS) in skeletal muscle L6 cells. Methods L6 cells were induced to differentiate into 6 groups, with 5 mmol / L glucose as the control group. The mixed FFA supplemented with 0.3 or 1 mmol / L as the low-FFA or low FFA group and 25 mmol / L Glucose is a high glucose control group. The medium supplemented with 0.3 and 1 mmol / L of FFA is high glucose, low FFA or high glucose, high FFA group. The insulin sensitivity of L6 cells was verified by ~ 3H-D-glucose uptake test (with or without 100 nmol / L insulin at 37 ℃ for 30 min), and the changes of intracellular ROS content were observed by fluorescent probe H2DCFDA. Results In the 5 mmol / L glucose group, the basal glucose value was 22777.6 ± 6608.7 in the low glucose control group, 26027.5 ± 4085.7 in the low glucose and low FFA group, and 146805.1 ± 21099.4 in the low and high glucose FFA group. After insulin stimulation, the low glucose control group was 76586.5 ± 18450.1, the low glucose low FFA group was 43738 ± 9203.6, the low glucose high FFA group was 32050.6 ± 3362.3, which was significantly lower than the low glucose control group (P <0.01). In 25 mmol / L glucose group, the basal glucose uptake was 11793.8 ± 551.4, while in high glucose low FFA group, 11887.3 ± 2082.3 high glucose and high FFA group was 13886.1 ± 3872.9, with no significant difference (P> 0.05) (P <0.01). After insulin stimulation, the high glucose control group was 42798.8 ± 9441.5, the high glucose low FFA group was 23946.9 ± 3839.6, and the high glucose and high FFA group was 13886.1 ± 3872.9. The difference was significant (P <0.01). The study of intracellular ROS found that low glucose low FFA group was 2234.2 ± 477.2, low glucose high FFA group was 1969.0 ± 480.9, high glucose control group was 1969.0 ± 229.4, high glucose low FFA group was 2184.5 ± 734.2, high glucose and high FFA group Was 2571.3 ± 96.7, which could significantly increase the ROS in L6 cells compared with the low glucose control group (615.3 ± 244.3) (P <0.01). There was no significant difference among the groups except the control group (P> 0.05). Conclusion High glucose and high FFA can induce oxidative stress in L6 cells, and high glucose and high FFA are one of the important causes of insulin resistance in skeletal muscle cells.