本文選題：垂體 + 黃體生成素��； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2010年博士論文

【摘要】： 第一部分PGC-1α協(xié)同SF-1調(diào)節(jié)黃體生成素和醛固酮合成的研究 過(guò)氧化物酶體增殖激活受體γ共激活因子1α(PGC-1α)能共刺激多種轉(zhuǎn)錄因子從而調(diào)節(jié)能量代謝進(jìn)程。核受體類固醇激素生成因子(SF-1)在垂體、性腺、腎上腺等內(nèi)分泌組織中高表達(dá),轉(zhuǎn)錄調(diào)控多種關(guān)鍵基因,同時(shí)調(diào)節(jié)下丘腦-垂體-性腺軸多水平的激素合成。為探索PGC-1α在類固醇激素合成及垂體激素分泌中的作用,本課題以小鼠垂體促性腺αT3-1細(xì)胞為研究模型,過(guò)表達(dá)PGC-1α后,通過(guò)real-time PCR實(shí)驗(yàn)檢測(cè)到LHβ和αGSU在水mRNA平的表達(dá)量升高,又進(jìn)一步通過(guò)酶聯(lián)免疫吸附試驗(yàn)(ELISA)證明,PGC-1α刺激αT3-1細(xì)胞分泌的黃體生成素增加。分析LHp啟動(dòng)子序列,發(fā)現(xiàn)-127 bp～-119 bp位點(diǎn)存在一個(gè)SF-1結(jié)合元件突變?cè)撛?雙熒光報(bào)告實(shí)驗(yàn)證明SF-1和PGC-1α對(duì)LHβ啟動(dòng)子的刺激作GSE (gonadotrope-specific element),用全部消失。染色質(zhì)免疫沉淀實(shí)驗(yàn)證明PGC-1α與SF-1共定位在LHβ啟動(dòng)子區(qū)含GSE元件的區(qū)域,并促進(jìn)LHβ轉(zhuǎn)錄。為進(jìn)一步證明PGC-1α對(duì)SF-1的共刺激作用,分析了二者相互作用的分 子機(jī)制。哺乳動(dòng)物雙雜交實(shí)驗(yàn)和免疫共沉淀實(shí)驗(yàn)證明PGC-1α與SF-1能在體內(nèi)形成復(fù)合物,進(jìn)一步實(shí)驗(yàn)證明二者能直接相互作用。該作用GST pull-down發(fā)生在PGC-1α的N端1～180氨基酸區(qū)域(含LXXLL模體,142～146氨基酸)與SF-1 C端187～462氨基酸區(qū)之間。構(gòu)建不同長(zhǎng)度的SF-1基因表達(dá)片段發(fā)現(xiàn),只有當(dāng)SF-1的最適激活結(jié)構(gòu)域和AF-2結(jié)(proximal activation domain)構(gòu)域同時(shí)存在時(shí),PGC-1α和SF-1才能直接結(jié)合。腎上腺皮質(zhì)是最主要的類固醇激素合成部位,細(xì)胞色素P450家族成員在 此參與膽固醇代謝、類固醇激素合成的多個(gè)催化反應(yīng)。Cyp11b2基因編碼的蛋白質(zhì)產(chǎn)物負(fù)責(zé)催化醛固酮合成的最后三步反應(yīng),是醛固酮合成的限速酶。在小鼠腎上腺皮質(zhì)Y-1細(xì)胞中,real-time PCR實(shí)驗(yàn)證明,PGC-1α能促使Cyp11b2,Cyp11b1和P450scc基因在mRNA水平表達(dá)上調(diào),ELISA實(shí)驗(yàn)進(jìn)一步檢測(cè)到PGC-1α協(xié)同SF-1促進(jìn)細(xì)胞醛固酮的分泌。 另一方面,通過(guò)雙熒光報(bào)告實(shí)驗(yàn)檢測(cè)到PGC-1α與雌激素受體相關(guān)受體家族成員ERRα和ERRγ共同促進(jìn)SF-1轉(zhuǎn)錄,在過(guò)表達(dá)PGC-1α的αT3-1細(xì)胞和Y-1細(xì)胞中,SF-1在mRNA水平和蛋白質(zhì)水平的表達(dá)量均顯著提高。綜上所述,本課題揭示了PGC-1α在類固醇激素和促性腺激素合成中具有重要的調(diào)節(jié)作用,分析了其刺激激素合成的分子機(jī)制,提示PGC-1α可能通過(guò)SF-1調(diào)節(jié)性腺和腎上腺的發(fā)育以及性別分化。 第二部分PGC-1α協(xié)同ERRα調(diào)節(jié)葡萄糖激酶表達(dá)的研究 過(guò)氧化物酶體增殖激活受體γ共激活因子1α(PGC-1α)是細(xì)胞能量代謝的關(guān)鍵調(diào)節(jié)子。它通過(guò)共激活多種核受體和轉(zhuǎn)錄因子,調(diào)節(jié)適應(yīng)性產(chǎn)熱、肝糖異生、脂肪酸氧化及線粒體生物合成等生理進(jìn)程。葡萄糖激酶是一種存在于哺乳動(dòng)物肝臟和胰腺中分子量為50 KDa的蛋白質(zhì),也被稱為Ⅳ型己糖激酶,是糖酵解過(guò)程中第一個(gè)限速酶。它在調(diào)節(jié)肝臟葡萄糖利用中發(fā)揮重要的作用,并接受胰島素信號(hào)刺激。 本課題研究發(fā)現(xiàn),大鼠葡萄糖激酶(glucokinase,Gck)啟動(dòng)子區(qū)存在一個(gè)保守的雌激素受體相關(guān)受體的結(jié)合元件ERRE(-52 bp～-39 bp),雙熒光報(bào)告實(shí)驗(yàn)分析了截短和位點(diǎn)突變的Gck啟動(dòng)子,證明PGC-1α和ERRα能利用該元件刺激Gck啟動(dòng)子的活性。EMSA實(shí)驗(yàn)進(jìn)一步證明ERRα能在體外結(jié)合ERRE元件,ChIP實(shí)驗(yàn)則證實(shí)PGC-1α和ERRα共同定位在大鼠肝細(xì)胞Gck啟動(dòng)子含該元件的區(qū)域,表明PGC-1α和ERRα通過(guò)ERRE元件促進(jìn)大鼠肝臟Gck基因轉(zhuǎn)錄。大鼠肝原代細(xì)胞感染PGC-1α和ERRα腺病毒后,Gck基因在mRNA和蛋白質(zhì)水平表達(dá)量均明顯上調(diào)。同時(shí),Gck的催化活性大大增加,這表明PGC-1α和ERRα在調(diào)節(jié)葡萄糖代謝方面發(fā)揮著重要的作用。PGC-1α和ERRα對(duì)Gck的促進(jìn)作用還可以增強(qiáng)胰島素刺激的效應(yīng)。最后,引入ERRα特異的siRNA (siERRα)和拮抗劑XCT790抑制ERRα表達(dá)后,胰島素誘導(dǎo)的Gck表達(dá)受到影響,充分證明了ERRα介導(dǎo)了胰島素誘導(dǎo)的Gck表達(dá)。綜上所述,本研究發(fā)現(xiàn)了PGC-1α和ERRα對(duì)Gck的促進(jìn)作用及對(duì)肝臟葡萄糖代謝的影響,證明ERRα在胰島素刺激葡萄糖激酶通路中的作用,并可以促進(jìn)Ⅱ型糖尿病葡萄糖穩(wěn)態(tài)的形成。
[Abstract]:Part PGC-1 SF-1 synergistic effect of LH on the synthesis of luteinizing hormone and aldosterone
The peroxisome proliferator activated receptor gamma co activating factor 1 alpha (PGC-1 alpha) can stimulate a variety of transcription factors to regulate the process of energy metabolism. The nuclear receptor steroid generating factor (SF-1) is highly expressed in the pituitary, gonadal, adrenal and other endocrine tissues, and regulates multiple key genes in the transcription and regulation of the hypothalamus pituitary gonadal axis. Level of hormone synthesis. In order to explore the role of PGC-1 alpha in the synthesis of steroid hormones and the secretion of pituitary hormones, this subject uses the mouse pituitary gonadotropin alpha T3-1 cells as the research model. After overexpressing PGC-1 alpha, the expression of LH beta and alpha GSU in the water mRNA level is increased by real-time PCR test, and further through the enzyme linked immunosorbent assay (E). LISA) proved that PGC-1 alpha stimulated the increase of luteinizing hormone secreted by alpha T3-1 cells. Analysis of the LHp promoter sequence and the existence of a SF-1 binding element mutation in the -127 BP to -119 BP site, the double fluorescence report showed that SF-1 and PGC-1 alpha were all disappearing. The immunoprecipitation experiment showed that PGC-1 alpha and SF-1 Co located the region of the GSE element in the LH beta promoter region, and promoted the LH beta transcription. In order to further demonstrate the co stimulation of PGC-1 alpha to SF-1, the interaction of the two groups was analyzed.
Sub mechanism. Both mammalian two hybrid experiments and immunoprecipitation experiments show that PGC-1 alpha and SF-1 can form complex in the body. Further experiments show that the two can interact directly. The action GST pull-down occurs between the 1~180 amino acid regions of the N end of PGC-1 alpha (including LXXLL module, 142~146 amino acid) and the 187~462 amino acid region of SF-1 C terminal. The construction of SF-1 gene expression fragments of different lengths showed that only when the optimal active domain of SF-1 and the AF-2 junction (proximal activation domain) domain existed simultaneously, PGC-1 alpha and SF-1 were directly combined. The adrenal cortex is the main steroid hormone synthesis site, and the cytochrome P450 family members are
This is involved in cholesterol metabolism, the multiple catalytic reaction of the steroid hormone synthesis, the protein product encoded by the.Cyp11b2 gene, is responsible for the final three step reaction of aldosterone synthesis, which is a speed limiting enzyme in the aldosterone synthesis. In the Y-1 cells of the adrenal cortex of mice, the real-time PCR experiment proved that PGC-1 alpha could induce the Cyp11b2, Cyp11b1 and P450scc genes in M. The level of RNA was upregulated. ELISA test further detected that PGC-1 alpha synergy SF-1 promoted aldosterone secretion.
On the other hand, PGC-1 alpha and estrogen receptor related receptor family members ERR alpha and ERR gamma were detected by double fluorescence report experiments to promote SF-1 transcription. The expression of SF-1 at mRNA level and protein level was significantly increased in PGC-1 alpha alpha T3-1 cells and Y-1 cells. To sum up, the subject revealed that PGC-1 a is steroid in steroid. The regulation of hormone and gonadotropin synthesis is important, and the molecular mechanism of its stimulatory hormone synthesis is analyzed. It is suggested that PGC-1 alpha may regulate the development of gonadal and adrenal glands and sex differentiation through SF-1.
The second part is to study the regulation of glucokinase expression by PGC-1 alpha and ERR alpha.
Peroxisome proliferator activated receptor gamma CO activation factor 1 alpha (PGC-1 alpha) is a key regulator of cell energy metabolism. It regulates physiological processes such as adaptive heat production, liver sugar isogenesis, fatty acid oxidation and mitochondrial biosynthesis by activating a variety of nuclear receptors and transcription factors. The protein of 50 KDa in the pancreas, also known as type IV hexokinase, is the first speed limiting enzyme in glycolysis. It plays an important role in regulating glucose utilization in the liver and is stimulated by insulin signal.
The study found that there is a conserved estrogen receptor related receptor binding element ERRE (-52 BP to -39 BP) in the promoter region of the glucokinase (Gck) promoter of the rat. The double fluorescence report experiment analyses the Gck promoter of the truncated and loci mutation, proving that PGC-1 alpha and ERR alpha can use this element to stimulate the active.EMSA solid of the Gck promoter. The test further demonstrated that ERR alpha could be combined with ERRE elements in vitro, and the ChIP experiment confirmed that PGC-1 A and ERR alpha were Co located in the Gck promoter region of rat hepatocytes, indicating that PGC-1 A and ERR a promoted the transcription of Gck genes in rat liver through ERRE components. The level of white matter expression is obviously up. Meanwhile, the catalytic activity of Gck is greatly increased, which indicates that PGC-1 alpha and ERR alpha play an important role in regulating glucose metabolism..PGC-1 A and ERR a can enhance the effect of Gck stimulation. Finally, the specific siRNA (siERR alpha) and antagonist XCT790 inhibition ERR are introduced. After the expression of alpha, the expression of insulin induced Gck was affected, which fully demonstrated that ERR a mediated the expression of Gck induced by insulin. To sum up, this study found that PGC-1 A and ERR alpha have a promoting effect on Gck and the effect on glucose metabolism in the liver. It is proved that ERR a plays a role in insulin stimulation of glucose kinase pathway and can promote type II. The formation of glucose homeostasis in diabetes.

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Primary hepatocyte culture in collagen gel mixture and collagen sandwich[J];World Journal of Gastroenterology;2004年05期

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本文編號(hào)：1848554