CAJ | 학술논문

转染促甲状腺素受体(TSHR) shRNA的3T3-L1脂肪细胞以牛促甲状腺素(TSH)刺激,酶联免疫吸附法(ELISA)检测培养基中肿瘤坏死因子α(TNF-α)水平,Western印迹法检测胰岛素受体底物1(IRS-1)的蛋白表达,免疫沉淀法检测IRS-1酪氨酸磷酸化.结果显示,1 mIU/ml TSH明显增加3T3-L1脂肪细胞TNF-α分泌[(341.85±12.00对522.67±36.22)ng/L,P＜0.01];随着TSH刺激浓度的增加,IRS-1蛋白表达及其酪氨酸磷酸化明显降低(均P＜0.01),而以RNA干扰下调TSHR后上述作用消失.另外,TNF-α拮抗剂WP9QY也可逆转TSH对IRS-1的下调作用.这些结果提示TSH与3T3-L1脂肪细胞表面TSHR结合后,可能通过促进TNF-α分泌进而下调IRS-1的表达及其酪氨酸磷酸化,而参与胰岛素抵抗的发生.
전염촉갑상선소수체(TSHR) shRNA적3T3-L1지방세포이우촉갑상선소(TSH)자격,매련면역흡부법(ELISA)검측배양기중종류배사인자α(TNF-α)수평,Western인적법검측이도소수체저물1(IRS-1)적단백표체,면역침정법검측IRS-1락안산린산화.결과현시,1 mIU/ml TSH명현증가3T3-L1지방세포TNF-α분비[(341.85±12.00대522.67±36.22)ng/L,P＜0.01];수착TSH자격농도적증가,IRS-1단백표체급기락안산린산화명현강저(균P＜0.01),이이RNA간우하조TSHR후상술작용소실.령외,TNF-α길항제WP9QY야가역전TSH대IRS-1적하조작용.저사결과제시TSH여3T3-L1지방세포표면TSHR결합후,가능통과촉진TNF-α분비진이하조IRS-1적표체급기락안산린산화,이삼여이도소저항적발생.
3T3-L1 adipocytes transfected with TSH receptor (TSHR) shRNA were incubated with bovine TSH.The concentration of tumor necrosis factor (TNF)-α in culture medium was measured by enzyme linked immunosorbent asssy.Protein level of insulin receptor substrate 1 (IRS-1) was quantified by Western blotting.Tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation.The results showed that 1 mIU/ml TSH significantly sitmulated TNF-α release in 3T3-L1 adipocytes [(341.85 ± 12.00 vs 522.67 ± 36.22) ng/L,P＜0.01],along with the decreases in IRS-1 protein expression and its tyrosine phosphorylation (P＜ 0.01).These effects disappeared when TSHR expression was down-regulated with RNA interference in 3T3-L1 adipocytes.In addition,WP9QY,a TNF-α antagonist,blocked TSH-decreased IRS-1 expresssion.These results suggest that TSH downregulates IRS-1 protein expression and its tyrosine phosphorylation through stimulating production of TNF-α,and thus contributes to the development of insulin resistance.