STAT3介导IL-6对细胞自噬抑制作用的研究

发布时间：2018-06-20 07:23

本文选题：自噬 + IL-6　；参考：《浙江大学》2014年硕士论文

【摘要】：目的：探讨IL-6对饥饿诱导的细胞自噬过程的影响及其分子机制。方法：将实验分为对照组、饥饿组及饥饿+IL-6组,首先采用western blot法检测了LC3蛋白的表达水平,细胞免疫荧光法统计LC3荧光点的量并借助电镜观察细胞内自噬泡数量变化和形态特征；western blot法检测并筛选自噬及IL-6下游主要通路蛋白表达水平；然后,采用关键信号分子的抑制剂并构建其突变体等方法,进一步检测其在IL-6抑制自噬中的作用,同时通过Beclin1siRNA, Atg14siRNA的干扰实验分析IL-6影响自噬过程的分子机制。结果：(1)我们发现,在饥饿处理的U937细胞内,外源IL-6(30ng/ml)的添加降低了LC3-9Ⅱ/LC3-Ⅰ比值,且二者的比值随IL-6浓度的增加而减小,同时IL-6的处理下调了免疫荧光反应中LC3的荧光点数,表明IL-6负向调控细胞自噬过程。(2)饥饿诱导下,细胞内与自噬相关的主要信号分子Akt, ERK1/2, STAT3的磷酸化水平均发生变化。IL-6(30ng/ml)添加后,Akt, ERK1/2的磷酸化水平未发生明显变化,但STAT3的磷酸化水平明显升高,活化了胞内STAT3通路,并促进了Bcl-2的表达,同时胞内自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值显著下调,p62未发生明显降解。(3) STAT3抑制剂LLL12作用于细胞后,P-STAT3活性显著降低,自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ, Beclin1及VPS34蛋白水平均有明显的上调；突变体STAT3Y705F的转染实验表明,IL-6的处理不影响STAT3Y705F转染的细胞内的自噬水平。(4)在Beclin1siRNA, Atg14siRNA转染的细胞中,IL-6的添加不影响饥饿作用后胞内自噬相关蛋白的表达水平。结论：综上所述,(1)IL-6抑制细胞自噬,STA3T介导IL-6抑制细胞自噬过程；(2)Beclin1, Atg14参与IL-6抑制细胞的自噬过程。深入探讨IL-6与细胞自噬的关系,将有助于探究与IL-6密切相关疾病的致病机制。
[Abstract]:Objective: To investigate the effect of IL-6 on the process of starvation induced autophagy and its molecular mechanism.
Methods: the experiment was divided into the control group, the starving group and the starving +IL-6 group. First, the expression level of LC3 protein was detected by the Western blot method. The amount of LC3 fluorescence point was measured by cell immunofluorescence and the changes of the number of autophagic vesicles and the morphological characteristics were observed by the electron microscope. The autophagy and the main pathway of the lower reaches of IL-6 were detected and screened by Western blot method. The protein expression level; then, using the inhibitors of key signal molecules and the construction of its mutants to further detect its role in the inhibition of autophagy by IL-6, and the molecular mechanism of IL-6 affecting autophagy through the interference experiments of Beclin1siRNA and Atg14siRNA.
Results: (1) we found that in the U937 cells treated with starvation, the addition of exogenous IL-6 (30ng/ml) reduced the ratio of LC3-9 II /LC3- I, and the ratio of the two decreased with the increase of IL-6 concentration, and the IL-6 treatment reduced the number of LC3 in the immunofluorescence reaction, indicating that the IL-6 negative direction regulates the autophagy process of the cells. (2) starvation induced. The phosphorylation of the main signal molecules associated with autophagy, Akt, ERK1/2, STAT3, was changed by.IL-6 (30ng/ml), and the phosphorylation level of Akt and ERK1/2 did not change obviously, but the phosphorylation level of STAT3 increased significantly, activated the intracellular STAT3 pathway, and promoted the expression of Bcl-2, and the intracellular autophagy related protein LC3- II. LC3- I ratio decreased significantly, and p62 did not degrade obviously. (3) the activity of P-STAT3 was significantly reduced after the action of STAT3 inhibitor LLL12 on the cells. The levels of the autophagy related protein LC3- II /LC3- I, Beclin1 and VPS34 protein were obviously up-regulated; the mutant STAT3Y705F transfection test showed that IL-6 treatment did not affect the cells transfected by STAT3Y705F. Autophagy level. (4) in Beclin1siRNA, Atg14siRNA transfected cells, the addition of IL-6 did not affect the expression level of autophagy related proteins after starvation.
Conclusion: (1) IL-6 inhibits autophagy, STA3T mediates IL-6 inhibition of autophagy, and (2) Beclin1, Atg14 participates in the autophagy process of IL-6 suppressor cells. The in-depth study of the relationship between IL-6 and cell autophagy will help to explore the pathogenesis of the closely related diseases associated with IL-6.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392.12

【共引文献】