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目的 :用TNF -α培养离体红细胞 ,观察其对胰岛素诱导的 β亚基自身磷酸化的影响 ,探讨TNF -α致胰岛素抵抗的分子机理。方法 :红细胞加或不加TNF -α(5ng/ml)培养 2 0h ,收集培养的红细胞 ,制备膜蛋白 ,并测定胰岛素诱导的受体自身磷酸化程度。结果 :红细胞TNF -α(- )培养 2 0h后 ,正常人红细胞胰岛素受体自身磷酸化程度为 80 .85± 4 3.92△A/mg蛋白 ,Ⅱ型糖尿病患者胰岛素受体的自身磷酸化程度为 2 4 .2 4± 8.0 5△A/m9蛋白 ,二者有显著性差异 (P <0 .0 5 )。 5ng/mlTNF -d培养 2 0h后 ,正常人红细胞胰岛素受体的磷酸化程度下降至 2 2 .95± 14 .93△A/mg蛋白 ,糖尿病组的胰岛素受体磷酸化程度下降至 8.81± 8.31△A/mg蛋白 ,与TNF -α(- )组相比 ,差异有显著性 (P <0 .0 5 )。正常人红细胞TNF -α(+)培养 2 0h后 ,其受体磷酸化程度与糖尿病组TNF -α(- )培养 2 0h后的受体磷酸化程度相比 ,二者差异无显著性。结论 :小剂量TNF-α(5ng/ml)培养红细胞 2 0h后 ,可抑制细胞膜上胰岛素受体的自身磷酸化 ,且正常人红细胞经TNF -α处理后 ,膜上胰岛素受体自身磷酸化程度可降至患者水平 ,这可能是TNF -α引起糖尿病病人对胰岛素抵抗的机制。
OBJECTIVE: To study the effect of TNF-α on the autophosphorylation of β subunit and to explore the molecular mechanism of TNF-α-induced insulin resistance. Methods: Erythrocytes were incubated with or without TNF-α (5ng / ml) for 20 h. The cultured erythrocytes were collected and membrane proteins were prepared. The extent of insulin-induced autophosphorylation was measured. Results: The level of autophosphorylation of erythrocyte insulin receptor was 80.85 ± 4. 3.92 △ A / mg protein after 20 h incubation with erythrocyte TNF-α (-). The degree of autophosphorylation of insulin receptor in type Ⅱ diabetes mellitus was There was a significant difference between the two groups (P <0.05). The level of phosphorylation of insulin receptor in erythrocytes decreased to 2.295 ± 14.93 △ A / mg protein after 20 h incubation with 5ng / ml TNF-α, and the phosphorylation of insulin receptor in diabetic group decreased to 8.81 ± 8.31 △ A / mg protein, compared with TNF-α (-) group, the difference was significant (P <0.05). Compared with normal control group, the level of phosphorylation of receptor of TNF-α (+) in normal human erythrocytes incubated with TNF-α (+) for 20 h was not significantly different between the two groups. CONCLUSION: Erythrocytes incubated with low dose of TNF-α (5ng / ml) for 20h can inhibit autophosphorylation of insulin receptor on the cell membrane, and the autophosphorylation of insulin receptor on the membrane of normal human erythrocytes after treatment with TNF- Can be reduced to the level of patients, which may be TNF-α-induced insulin resistance in diabetic patients.