本文選題：PGC-1β　切入點：NRF-1　出處：《中國協(xié)和醫(yī)科大學》2010年博士論文　論文類型：學位論文

【摘要】： 第一部分 NRF-1和ERRa是介導PGC-1β調(diào)節(jié)小鼠C2C12細胞線粒體生物合成中的重要轉(zhuǎn)錄因子 PPAR-y共激活因子-1β(PGC-1β)是PPAR-y共激活因子家族成員之一,與PGC-1α有高度同源性。目前發(fā)現(xiàn),PGC-1β是調(diào)節(jié)線粒體脂肪酸p氧化和氧化磷酸化的非常重要的轉(zhuǎn)錄因子。但是現(xiàn)在對PGC-1β作用機制研究的還不夠清楚。我們發(fā)現(xiàn)在小鼠肌管細胞發(fā)生過程中PGC-1β的表達明顯增加,并且利用siRNA干擾內(nèi)源PGC-1β表達后,線粒體相關基因的表達也相應減少。因此,可以看出PGC-1β在小鼠肌管細胞線粒體生物合成中發(fā)揮重要的調(diào)控作用。進一步研究發(fā)現(xiàn),PGC-1β可以激活線粒體相關目標基因的表達,例如：細胞色素C, ATP合成酶5β和ALAS-1。PGC-1β激活這些基因是通過與至少兩個轉(zhuǎn)錄因子NRF-1和ERRα完成的。我們進一步證明了在體內(nèi)外PGC-1β和NRF-1存在相互作用。通過雙熒光報告基因?qū)嶒灠l(fā)現(xiàn),在這些基因的啟動子上對NRF-1和ERRα結(jié)合元件進行突變和截短后,減弱了PGC-1β對基因的激活作用。另外,利用siRNA對NRF-1和ERRα進行干擾后,PGC-1β對以上基因及其他線粒體相關基因mRNA水平的激活作用減弱,并且,線粒體的拷貝數(shù)及細胞呼吸作用也減弱。因此,可以得出結(jié)論,PGC-1β是通過與NRF-1和ERRα相互作用對線粒體的生物合成和相關功能進行調(diào)節(jié)的。 第二部分 NRF-1介導PGC1β促進ALAS-1表達而調(diào)控血紅素生成 亞鐵血紅素是許多血紅素相關蛋白的重要組成成分,由鐵原子及原卟啉區(qū)組成。它的功能包括氧的運輸、能量代謝和藥物的生物轉(zhuǎn)化,亞鐵血紅素對于哺乳動物的生命是必須的。5-氨基乙酰丙酸合成酶1 (5-aminolevulinate synthase, ALAS-1)為線粒體基質(zhì)酶,催化亞鐵血紅素合成的第一產(chǎn)物合成。本研究發(fā)現(xiàn),肝臟中的ALAS-1受過氧化物酶體增生物激活受體共激活子1β(peroxisome proliferator-activated receptorycoactivatorlβ, PGC-1β)調(diào)節(jié),PGC-1β通過NRF-1介導促進ALAS-1基因表達。對ALAS-1啟動子NRF-1結(jié)合元件進行突變發(fā)現(xiàn)PGC-1β激活ALAS-1啟動子轉(zhuǎn)錄的作用減弱,我們同時通過干擾內(nèi)源性NRF-1表達導致PGC-1β促進ALAS-1基因表達功能減弱進一步證明NRF-1是介導PGC-1β促進ALAS-1基因表達的轉(zhuǎn)錄因子。本研究還檢測參與亞鐵血紅素合成過程中的其他七種酶,發(fā)現(xiàn)PGC-1β可以促進ALAD (5-aminolevulinate dehydratase)、HMBS (hydroxymethylbilane synthase), UROD (uroporphyrinogen decarboxylase),. CPOX (coproporphyrinogen oxidase)、PPOX (protoporphyrinogen oxidase)、FECH (ferrochelatase)的mRNA水平表達。同時,干擾NRF-1后這幾種基因表達均下調(diào)。實驗結(jié)果顯示,PGC-1β可以促進催化亞鐵血紅素合成的相關酶的表達,PGC-1β是調(diào)節(jié)肝臟亞鐵血紅素合成的重要轉(zhuǎn)錄因子。
[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 偽. 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 偽 binding elements on the promoters of these genes weakened the activation of PGC-1 尾. After interfering with NRF-1 and ERR 偽 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 偽. 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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R363

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本文編號：1641889