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目的探讨2型糖尿病(T2DM)患者脂肪细胞内异常胰岛素信号转导与核因子(NF)-κB活化的关系;研究NF-κB靶向诱捕分子(NF-κBdecoy)在体外对胰岛素抵抗的作用。方法取T2DM患者及非糖尿病患者腹部皮下脂肪组织进行原代培养,用免疫沉淀法及蛋白质印迹法检测两组脂肪细胞内胰岛素刺激后胰岛素信号转导分子胰岛素受体底物(IRS)-1酪氨酸磷酸化及Akt-Ser473磷酸化程度,用电泳迁移率变动分析(EMSA)测定两组脂肪细胞内NF-κB的活性;脂质体瞬时转染法将NF-κBdecoy分子转入T2DM患者脂肪细胞内,再检测转染后上述胰岛素信号分子及NF-κB的活性。结果T2DM患者脂肪细胞内IRS-1酪氨酸磷酸化及Akt-Ser473磷酸化水平明显低于非糖尿病患者(P<0.05),NF-κB的活性明显高于非糖尿病患者(P<0.01);转染NF-κBdecoy分子后T2DM患者脂肪细胞内NF-κB的活性较转染前明显降低(P<0.05),IRS-1酪氨酸磷酸化及Akt-Ser473磷酸化水平较转染前有明显升高(P<0.05)。结论T2DM患者的腹部皮下脂肪细胞存在胰岛素抵抗(IR)和NF-κB过度活化;NF-κB靶向诱捕分子体外能部分逆转胰岛素抵抗作用。
Objective To investigate the relationship between abnormal insulin signal transduction and nuclear factor (NF) -κB activation in type 2 diabetes mellitus (T2DM) patients and the effect of NF-κB targeting decoy molecule (NF-κB) on insulin resistance in vitro. Methods Subcutaneous adipose tissue from T2DM patients and non-diabetic patients were cultured in primary culture. Insulin-stimulated insulin signaling molecule insulin receptor substrate (IRS) -1 was assayed by immunoprecipitation and Western blotting in both groups. The phosphorylation of Akt-Ser473 and phosphorylation of Akt-Ser473 were detected by enzyme-linked immunosorbent assay (ELISA). The activity of NF-κB in adipocytes was measured by electrophoretic mobility shift assay (EMSA) Cells, and then detect the transfection of the above insulin signaling molecules and NF-κB activity. Results The levels of IRS-1 tyrosine phosphorylation and phosphorylation of Akt-Ser473 in T2DM patients were significantly lower than those in non-diabetic patients (P <0.05). The activity of NF-κB in T2DM patients was significantly higher than that in non-diabetic patients (P <0.01). The activity of NF-κB in adipocytes of T2DM patients after transfection with NF-κBdecoy was significantly lower than that before transfection (P <0.05). The levels of tyrosine phosphorylation of IRS-1 and phosphorylation of Akt-Ser473 were significantly higher than those before transfection Increased (P <0.05). CONCLUSIONS: Insulin resistance (IR) and NF-κB overexpression are present in subcutaneous adipocytes of T2DM patients. NF-κB targeting decoy molecules can partially reverse insulin resistance in vitro.