自噬在脂肪细胞慢性炎症反应中的调节作用及雷帕霉素的抗炎机制研究

发布时间：2018-05-08 06:26

本文选题：脂肪细胞 + 自噬　；参考：《上海交通大学》2015年博士论文

【摘要】：第一部分脂肪细胞应激诱导自噬的作用及机制研究目的:探讨高脂环境下自噬活性的变化及其与内质网应激的关系。方法:通过Western blot、免疫荧光、电镜三种方法检测棕榈酸负荷下分化成熟脂肪细胞自噬活性的变化;RT-PCR和Western blot方法检测内质网应激信号通路,观察自噬与内质网应激发生的先后顺序,并通过抑制内质网应激信号通路观察自噬水平的变化,明确自噬的诱导机制;通过药物性抑制自噬,分别用Western blot方法检测对细胞内质网应激信号及MTT法检测对细胞存活率的影响。结果:棕榈酸显著诱导分化成熟的脂肪细胞自噬活性的增加,表现为自噬体和自噬溶酶体形成增多,自噬特异性蛋白LC3-II的积累量增多及自噬流的增加;棕榈酸诱导内质网应激的发生且先于自噬的激活,并且抑制内质网应激导致LC3-II的积累量降低;此外,抑制自噬增加了细胞对应激和凋亡的敏感性。结论:棕榈酸激活成熟分化脂肪细胞的自噬活性增加;自噬的激活部分依赖棕榈酸诱导的内质网应激途径;自噬作为适应性反应在应激环境下增加细胞对内质网应激和细胞调亡的抵抗。第二部分自噬对脂肪细胞的炎症因子的调节作用目的:探讨自噬激活对脂肪细胞炎症因子的调节作用方法:通过免疫组化和RT-PCR方法,观察饮食诱导肥胖鼠脂肪组织及高脂诱导脂肪细胞的炎症表现以及自噬相关基因的表达,明确炎症和自噬的水平;分别通过药物性抑制和si RNA干扰自噬基因的方法阻断自噬,RT-PCR方法检测炎症因子MCP-1和IL-6的表达水平,明确自噬对炎症因子的调节作用。结果:饮食诱导肥胖鼠脂肪组织切片的HE染色和CD68免疫组化分析结果表现为炎症细胞的浸润增加,RT-PCR方法检测棕榈酸诱导分化脂肪细胞炎症因子的表达水平上调。肥胖诱导的脂肪组织及棕榈酸诱导脂肪细胞都表现为自噬基因的表达升高。3-MA自噬特异性抑制和si RNA转染自噬相关基因Becn1干扰自噬的形成,RT-PCR法检测MCP-1和IL-6的表达水平上调。结论:在肥胖诱导的脂肪组织和单独脂肪细胞的炎症反应中自噬被激活,自噬作为应激环境下的适应性机制参与炎症反应的调节,并发挥抗炎的作用。第三部分雷帕霉素的抗应激和抗炎作用及其与自噬活性的关系目的:探讨雷帕霉素的抗应激和抗炎作用及其与自噬活性的关系方法:Western blot方法检测m TORC1信号下游磷酸化S6K1蛋白表达,观察棕榈酸对m TORC1信号的影响,同时验证雷帕霉素对脂肪细胞m TORC1信号的抑制作用。分别用雷帕霉素预处理脂肪细胞,通过Western blot和RT-PCR方法检测雷帕霉素对内质网应激信号蛋白和炎症因子及炎症通路NF-κB表达的影响;并通过药物性抑制和si RNA干扰自噬的方法进一步明确,雷帕霉素对内质网应激和炎症反应的作用与自噬的关系。结果:棕榈酸显著诱导脂肪细胞磷酸化S6K1蛋白表达增加,且可被雷帕霉素所抑制;雷帕霉素显著下调棕榈酸诱导的内质网应激信号蛋白的表达,但CQ抑制自噬消除了雷帕霉素的抗应激作用;雷帕霉素显著抑制NF-κB炎症信号及其介导的炎症因子MCP-1和IL-6的表达,si Becn1干扰自噬可部分阻断雷帕霉素的抗炎作用。结论:棕榈酸诱导m TORC1信号上调促进炎症反应的发生,雷帕霉素发挥抗内质网应激和抗炎的作用,且雷帕霉素的这些有益作用部分依赖自噬途径的激活。
[Abstract]:The first part of the study on the role and mechanism of autophagy induced by adipocyte stress: To explore the changes of autophagic activity and the relationship with endoplasmic reticulum stress in high fat environment. Methods: the changes of autophagic activity of mature fat cells under palmitic acid load were detected by three methods of Western blot, immunofluorescence and electron microscopy; RT-PCR and Western blot Methods to detect the endoplasmic reticulum stress signal pathway, observe the sequence of autophagy and endoplasmic reticulum stress, and to observe the changes of autophagy by inhibiting the endoplasmic reticulum stress signal pathway and clear the induction mechanism of autophagy. Through the drug inhibition of autophagy, the Western blot method was used to detect the endoplasmic reticulum stress signal and the MTT method, respectively. Results: palmitic acid significantly induced an increase in autophagic activity of mature adipocytes, showing an increase in the formation of autophagosomes and autophagosomes, increased accumulation of autophagic specific protein LC3-II and increased autophagic flow; palmitic acid induced endoplasmic reticulum activation and inhibition of autophagic activation, and inhibition of endoplasmic reticulum. In addition, inhibition of autophagy increases the sensitivity of LC3-II to stress and apoptosis. Conclusion: palmitic acid activates the autophagic activity of mature and differentiated adipocytes, the activation of autophagy is partly dependent on the endoplasmic reticulum stress pathway induced by palmitic acid; autophagy increases in the stress environment as an adaptive response. The resistance of cell to endoplasmic reticulum stress and cell apoptosis. Second the regulation of autophagy to the inflammatory factors of adipocytes: Objective To investigate the regulation of autophagy activation on adipocyte inflammatory factors: the inflammatory expression of fat and high fat induced adipose cells in obese rats was observed by immunohistochemical and RT-PCR methods. And the expression of autophagy related genes, clear the level of inflammation and autophagy; blocking autophagy by drug inhibition and Si RNA interfering autophagy genes, RT-PCR methods to detect the expression level of inflammatory factors MCP-1 and IL-6, and the regulation of autophagy to inflammatory factors. Fruit: HE staining in fat tissue slices of obese rats induced by diet The results of CD68 immunohistochemical analysis showed that the infiltration of inflammatory cells increased, and the expression of palmitic acid induced the expression level of inflammatory factors in differentiated adipocytes was up-regulated by RT-PCR method. Adipose tissue and palmitic acid induced adipocytes were expressed as autophagic gene expression increased by.3-MA autophagy specific inhibition and Si RNA transfected autophagy The autophagy Becn1 interferes with the formation of autophagy, and the expression level of MCP-1 and IL-6 is up regulated by the RT-PCR method. Conclusion: autophagy is activated in the inflammatory response of fat induced adipose tissue and individual adipocytes. Autophagy participates in the regulation of inflammatory response as an adaptive mechanism under stress environment and plays an anti-inflammatory effect. Third part rapamycin The anti stress and anti-inflammatory effects and its relationship with autophagy: To explore the anti stress and anti-inflammatory effects of rapamycin and its relationship with autophagy: Western blot method was used to detect the expression of S6K1 protein in the downstream of M TORC1 signal, and to observe the effect of palmitic acid on M TORC1 signal, and to verify that rapamycin on M TO in adipocytes. The inhibitory effects of RC1 signal were pretreated with rapamycin. The effects of rapamycin on endoplasmic reticulum stress signal protein and inflammatory factors and the expression of NF- kappa B in endoplasmic reticulum were detected by rapamycin and Western blot and RT-PCR, and the methods of drug inhibition and Si RNA to interfere with autophagy were further clarified. Rapamycin should be used for endoplasmic reticulum. Results: palmitic acid significantly induced the increased expression of phosphorylated S6K1 protein in adipocytes and inhibited by rapamycin, and rapamycin significantly downregulated the expression of endoplasmic reticulum stress signal protein induced by palmitic acid, but CQ inhibited autophagy to eliminate the anti stress effect of rapamycin; rapamycin Significant inhibition of NF- kappa B inflammatory signal and its mediated expression of inflammatory factors MCP-1 and IL-6, Si Becn1 interference with autophagy can partially block the anti-inflammatory effects of rapamycin. Conclusion: the up-regulated m TORC1 signal induced by palmitic acid promotes the occurrence of inflammatory response, rapamycin plays the role of anti endoplasmic reticulum stress and anti-inflammatory, and the beneficial effects of rapamycin The activation of autophagy is partly dependent on the autophagy pathway.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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