【作者】 余晓岚；

【导师】 杨在清；

【作者基本信息】 华中农业大学 ， 生物工程， 2013， 硕士

【摘要】 抵抗素(resistin)是机体内非常重要的一类脂肪细胞因子,可作用于脂肪,肝脏,骨骼肌,并与胰岛素抵抗,炎症反应密切相关,影响糖脂代谢,并参与机体的能量调节。线粒体是机体内进行三羧酸循环及脂肪氧化磷酸化的重要场所,与能量代谢密切相关。近些年的研究表明,线粒体功能在肥胖,糖尿病以及胰岛素抵抗的个体中均受到损伤,甚至有线粒体数量减少的现象出现。已有研究表明resistin可以显著下调线粒体的数量,并且呈现浓度依赖性,并且影响脂肪酸氧化,促进脂肪积累。但是resistin的受体以及下调线粒体数量的具体途径并不清楚。本研究采用RT-PCR, Western Blot,双荧光报告系统和细胞转染等实验技术,研究resistin作用的受体及下调线粒体数量的信号通路。结果如下：1.在HepG2细胞中,用TLR4抗体封闭TLR4后,resistin仍可以下调线粒体数量,而用Decorin的活性抑制剂处理细胞,resistin也仍然可以下调线粒体,而基因芯片结果表明ROR1在resistin处理的hepG2细胞中不表达,表明resistin在hepG2细胞中下调线粒体的作用不是通过这三个受体。2. Resistin刺激hepG2细胞后,cAMP水平得到显著上升,而cGMP水平没有明显变化。通过先关抑制剂实验表明resistin通过激活蛋白激酶C(PKC),从而激活蛋白激酶G(PKG),且不依赖于cGMP信号途径。3.通过基因芯片技术及RT-PCR实验,证明resistin通过NF-κB信号途径发挥作用。超表达p65可以下调线粒体的数量,而用RNAi技术干扰p65则可以挽救线粒体数量被resistin下调的现象。此外,通过构建p65点突变载体和抑制剂实验表明,p65可以被PKG磷酸化激活,其磷酸化位点可能是p65的Thr464位点。4.在HEK293细胞中超表达p65,则发现pgc1α的表达量下降；而用RNAi技术干扰p65,则发现pgc1α的表达量上升；并且超表达PGC1α可以上调HEK293细胞中线粒体的含量。通过PLA实验证明p65和PGC1α是相互结合的,并且resistin促进其结合,而PKG的抑制剂KT5823则抑制两者结合。研究还发现resistin抑制PGC1α启动子活性,并且呈现浓度依赖性,而p65则通过与PGC1α结合而抑制PGC1α的自激活,从而抑制启动子的活性。5.运用RNAi技术,RT-PCR等实验探索resistin作用于PKC上游的信号通路。实验表明核转录因子p62被敲减后,可以阻碍resistin下调线粒体数量。此外,resistin下调线粒体可能是通过TNFR1发挥作用。更多还原

【Abstract】 Resistin is an important type of adipokine, which can effect adipose tissue, liver and skeletal muscle. Besides, it is closely related with insulin signaling pathways and inflammatory reaction by influencing glucose and fat metabolism.Consequently, it is involved in the regulation of energy metabolism.Mitochondria are important places for tricarboxylic acid cycle and fat oxidative phosphorylation, which are closely related with energy metabolism. Recent studies have demonstrated that the function of mitochondria was damaged in obese, diabetic people and the ones who had been accompanied with insulin resistance, besides, the content of mitochondria were also diminished in some obeses people.Based on the preview results, we have proved that resistin downregulated the number of mitochondria by the time and dose-dependent and induced fat accumulation. However, the resistin’s receptors and the signaling pathways of resistin’s effects on mitochondria are still unclear. In our study, we investigated the resistin’s possible receptors and the signaling pathways of resistin on mitochondria in detail by using Realtime PCR, Western Blot, dual-luciferase reporter assay system and cell transfection and so on. At last, we got the following results:1. After using TLR4 antibody to incubate hepG2 cells, we found the content of mitochondria were still downregulated by resistin treatment. Besides, We found the content of mitochondria were still downregulated by resistin after using the decorin inhibitor to deal with hepG2 cells. And, the ROR1 was not expressed after resistin administration through gene chip analysis.Thus, we excluded that resistin effected on mitochondria through these three possible receptors in hepG2 cells.2. Resistin increased the level of cAMP, but did not affect the level of cGMP in the hepG2 cells. By the inhibitor treatment and RT-PCR, we proved that resistin activated the protein kinase G (PKG) by activating the protein kinase C (PKC) independent of cGMP.3. By using the gene chip technique and RT-PCR, we proved that resistin played an important role in the NF-kappa B signaling pathway. Overexpressing p65 could diminish mitochondrial content, however, by using RNAi technology to interfere p65 expression, we could block the resistin’s functions on mitochonria. In addition, we found that PKG may activate p65 through phosphorylating the Thr464 of p65.4. Overexpression of PGC1α could reverse the resistin’s effect on mitochondria. Besides, we found that overexpressing p65 could decrease the pgc1α expression and inhibiting p65 could increase pgcla expression, inversely. Besides, we proved that p65 and PGC1α were combined and competed with each other and resistin enhanced the interaction by PLA experiments. Then, we found resistin could inhibit the activity of pgc1α promoter in dose-dependent manner. And, p65 inhibited the activity of pgc1α promoter by the interaction with PGC1α, as a result, PGC1α could not coactive itself.5. By using RNAi technology, RT-PCR, we explored the possible factor involved in the upstream pathway of PKC. After inhibiting p62 by RNA interfence, we found it could block resistin’s effect on mitochondria. Besides, we found TNFR1 may participate in the sigaling pathway of resistin on mitochondria.更多还原