【摘要】 【目的】明确Src与胶原同源插头蛋白(Src homology and collagen homology,Shc)调控曲格列酮(troglitazone,TZ)引起的猪血管内皮(porcine aortic endothelial,PAE)细胞自噬的机制。【方法】我们先利用激光共聚焦显微镜、蛋白免疫杂交检测了TZ引起的PAE细胞自噬;然后通过siRNA干扰敲降Shc,转染wtShc、3mShc等质粒的方法确定了p52Shc参与自噬的调控;最后通过siRNA干扰敲降Ulk1得到最终结论。【结果】通过研究发现,敲降Shc,会增加细胞的自噬;而过量表达p52Shc抑制了TZ引起的细胞自噬;p52Shc抑制自噬与其自身的酪氨酸磷酸化位点Tyr239、Tyr240和Tyr317相关;同时发现,p52Shc能抑制自噬调节分子磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase,AMPK)及其下游底物Unc51样激酶1(UNC-51-like kinase-1,Ulk1)的活性。【结论】Shc通过调控AMPK与Ulk1的磷酸化调节TZ引起的细胞自噬。更多还原

【Abstract】 [Objective] We investigated the mechanism of Src homology and collagen homology( Shc) in autophagy caused by troglitazone( TZ). [Methods] To reveal the regulatory role of p52Shc in autophagy,we used confocal microscopy and immunoblotting to examine autophagy induced by TZ. Then we used small RNA interference( siRNA) to deplete Shc and plasmids transfection to overexpress wtShc as well as 3mShc in PAE cells. Finally,we reached conclusion by detecting autophagic status following the deprivation of UNC-51-like kinase-1( Ulk1) by siRNA. [Results]We found that the deprivation of Shc showed to enhance autophagy,whereas p52Shc over expression suppressed TZ-depended autophagy concurring with an attenuated AMP-activated protein kinase( AMPK) and Ulk1 signaling. Besides,it demonstrated that p52Shc tyrosine sites of 239,240 and 317 implemented a critical role in the process. [Conclusion]Collectively,Shc adaptor protein was involved in TZ-inducing autophagy likely via affecting AMPK and Ulk1 signaling.更多还原