PGC-1β调节小鼠C2C12细胞线粒体发生和大鼠肝细胞血红素合成的机制研究

发布时间：2018-03-21 02:38

本文选题：PGC-1β　切入点：NRF-1　出处：《中国协和医科大学》2010年博士论文　论文类型：学位论文

【摘要】： 第一部分 NRF-1和ERRa是介导PGC-1β调节小鼠C2C12细胞线粒体生物合成中的重要转录因子 PPAR-y共激活因子-1β(PGC-1β)是PPAR-y共激活因子家族成员之一,与PGC-1α有高度同源性。目前发现,PGC-1β是调节线粒体脂肪酸p氧化和氧化磷酸化的非常重要的转录因子。但是现在对PGC-1β作用机制研究的还不够清楚。我们发现在小鼠肌管细胞发生过程中PGC-1β的表达明显增加,并且利用siRNA干扰内源PGC-1β表达后,线粒体相关基因的表达也相应减少。因此,可以看出PGC-1β在小鼠肌管细胞线粒体生物合成中发挥重要的调控作用。进一步研究发现,PGC-1β可以激活线粒体相关目标基因的表达,例如：细胞色素C, ATP合成酶5β和ALAS-1。PGC-1β激活这些基因是通过与至少两个转录因子NRF-1和ERRα完成的。我们进一步证明了在体内外PGC-1β和NRF-1存在相互作用。通过双荧光报告基因实验发现,在这些基因的启动子上对NRF-1和ERRα结合元件进行突变和截短后,减弱了PGC-1β对基因的激活作用。另外,利用siRNA对NRF-1和ERRα进行干扰后,PGC-1β对以上基因及其他线粒体相关基因mRNA水平的激活作用减弱,并且,线粒体的拷贝数及细胞呼吸作用也减弱。因此,可以得出结论,PGC-1β是通过与NRF-1和ERRα相互作用对线粒体的生物合成和相关功能进行调节的。第二部分 NRF-1介导PGC1β促进ALAS-1表达而调控血红素生成亚铁血红素是许多血红素相关蛋白的重要组成成分,由铁原子及原卟啉区组成。它的功能包括氧的运输、能量代谢和药物的生物转化,亚铁血红素对于哺乳动物的生命是必须的。5-氨基乙酰丙酸合成酶1 (5-aminolevulinate synthase, ALAS-1)为线粒体基质酶,催化亚铁血红素合成的第一产物合成。本研究发现,肝脏中的ALAS-1受过氧化物酶体增生物激活受体共激活子1β(peroxisome proliferator-activated receptorycoactivatorlβ, PGC-1β)调节,PGC-1β通过NRF-1介导促进ALAS-1基因表达。对ALAS-1启动子NRF-1结合元件进行突变发现PGC-1β激活ALAS-1启动子转录的作用减弱,我们同时通过干扰内源性NRF-1表达导致PGC-1β促进ALAS-1基因表达功能减弱进一步证明NRF-1是介导PGC-1β促进ALAS-1基因表达的转录因子。本研究还检测参与亚铁血红素合成过程中的其他七种酶,发现PGC-1β可以促进ALAD (5-aminolevulinate dehydratase)、HMBS (hydroxymethylbilane synthase), UROD (uroporphyrinogen decarboxylase),. CPOX (coproporphyrinogen oxidase)、PPOX (protoporphyrinogen oxidase)、FECH (ferrochelatase)的mRNA水平表达。同时,干扰NRF-1后这几种基因表达均下调。实验结果显示,PGC-1β可以促进催化亚铁血红素合成的相关酶的表达,PGC-1β是调节肝脏亚铁血红素合成的重要转录因子。
[Abstract]:Part one. NRF-1 and ERRa are important transcription factors in regulating mitochondrial biosynthesis of mouse C2C12 cells mediated by PGC-1 尾. PPAR-y co-activator 1 尾 -PGC-1 尾 is a member of PPAR-y coactivator family. It is found that PGC-1 尾 is a very important transcription factor in regulating the oxidation and oxidative phosphorylation of mitochondrial fatty acids. However, the mechanism of PGC-1 尾 action is not clear enough. We found that in mouse muscle, PGC-1 尾 is a very important transcription factor in the regulation of mitochondrial fatty acid p oxidation and oxidative phosphorylation. The expression of PGC-1 尾 was significantly increased during the process of tubulogenesis. And when siRNA interfered with the expression of endogenous PGC-1 尾, the expression of mitochondrial related genes decreased. It can be seen that PGC-1 尾 plays an important role in the regulation of mitochondrial biosynthesis in mouse myotube cells. For example, cytochrome C, ATP synthase 5 尾 and ALAS-1.PGC-1 尾 activate these genes by interacting with at least two transcription factors, NRF-1 and ERR 伪. We further demonstrate the interaction between PGC-1 尾 and NRF-1 in vivo and in vitro. The mutation and truncation of NRF-1 and ERR 伪 binding elements on the promoters of these genes weakened the activation of PGC-1 尾. After interfering with NRF-1 and ERR 伪 by siRNA, PGC-1 尾 weakened the activation of mRNA level of these genes and other mtDNA related genes, and the copy number of mitochondria and cell respiration were also weakened. It is concluded that PGC-1 尾 regulates mitochondrial biosynthesis and related functions through interaction with NRF-1 and ERR 伪. Part two. NRF-1 mediates PGC1 尾 promotes ALAS-1 expression and regulates heme production. Ferroheme is an important component of many heme-associated proteins, consisting of iron atoms and protoporphyrin regions. Its functions include oxygen transport, energy metabolism and drug biotransformation. Heme is the first product of heme synthesis that catalyzes the synthesis of heme, which is necessary for mammalian life. 5 aminolevulinate synthase (ALAS-1) is a mitochondrial matrix enzyme. ALAS-1 in the liver was regulated by peroxisome proliferator-activated receptorycoactivatorl 尾 -PGC-1 尾). The ALAS-1 promoter NRF-1 binding element mutation revealed that PGC-1 尾 activated ALAS-1 promoter transcription. By interfering with endogenous NRF-1 expression, we further demonstrated that NRF-1 is a transcription factor that mediates PGC-1 尾 to promote ALAS-1 gene expression. We also detected seven other enzymes involved in heme synthesis. It was found that PGC-1 尾 could promote the expression of mRNA in ALAD 5 aminolevulinate dehydratase, UROD uroporphyrinogen decarboxylase, CPOX porporporphyrinogen oxidase PPOX protoporphyrinogen oxidase, and FECH ferrochelase. These genes were down-regulated after interfering with NRF-1. The results showed that PGC-1 尾 could promote the expression of related enzymes that catalyze heme synthesis. PGC-1 尾 is an important transcription factor regulating hepatic heme synthesis.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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