發(fā)布時(shí)間：2018-08-12 16:27

【摘要】：脂肪組織主要由脂肪細(xì)胞所組成,過多的熱量攝入并以甘油三酯的形式儲(chǔ)存在白色脂肪細(xì)胞內(nèi)是肥胖發(fā)生的關(guān)鍵。前脂肪細(xì)胞的增殖分化是成熟脂肪細(xì)胞形成的核心,從前脂肪細(xì)胞分化成成熟的脂肪細(xì)胞的過程十分復(fù)雜,特別是機(jī)體內(nèi)多種細(xì)胞因子和激素在脂肪細(xì)胞發(fā)生過程中可能都有調(diào)節(jié)作用。 BRD2屬于BET蛋白家族,在哺乳動(dòng)物細(xì)胞中廣泛表達(dá),具有多種生物學(xué)功能,最新的研究表明Brd2基因突變能導(dǎo)致小鼠過度肥胖而不引起糖尿病,但是其作用的分子機(jī)制并不明確。在本課題中,我們觀察了Brd2表達(dá)變化對(duì)前脂肪細(xì)胞向脂肪細(xì)胞分化過程的影響,并且重點(diǎn)研究了Brd2參與的信號(hào)通路在影響前脂肪細(xì)胞向脂肪細(xì)胞分化作用的分子機(jī)制,彌補(bǔ)了這一部分研究的空白。 在研究中,我們發(fā)現(xiàn)Brd2表達(dá)的下調(diào),能促使3T3-L1前脂肪細(xì)胞向脂肪細(xì)胞的分化,甚至在無任何誘導(dǎo)分化劑的情況下,部分細(xì)胞也能分化成脂肪細(xì)胞；相反的,當(dāng)Brd2過表達(dá)時(shí),即使誘導(dǎo)分化至第8天,細(xì)胞分化也幾乎完全被抑制。這就更加表明Brd2在前脂肪細(xì)胞向脂肪細(xì)胞分化過程中有重要的作用,同時(shí)我們認(rèn)為,上述現(xiàn)象的產(chǎn)生可能是由于Brd2表達(dá)的變化影響了一些分化相關(guān)基因甚至是關(guān)鍵基因的表達(dá)。 為了研究Brd2表達(dá)的變化對(duì)脂肪細(xì)胞分化相關(guān)基因表達(dá)的影響,一方面我們利用RNA干擾技術(shù),下調(diào)3T3-L1前脂肪細(xì)胞內(nèi)Brd2的表達(dá)水平；另一方面運(yùn)用將pmBrd2載體將Brd2基因?qū)?T3-L1前脂肪細(xì)胞,使細(xì)胞內(nèi)的Brd2過表達(dá),然后先通過RT-PCR技術(shù)檢測(cè)了PPARy和C/EBPa的時(shí)序性表達(dá),結(jié)果表明Brd2對(duì)脂肪細(xì)胞分化的兩個(gè)關(guān)鍵基因PPARy和C/EBPa都有顯著的負(fù)調(diào)節(jié)作用；繼而運(yùn)用Q-PCR技術(shù),我們又檢測(cè)了其它相關(guān)基因的表達(dá),結(jié)果顯示,422/aP2、Glut4、 Leptin和Irs-1的表達(dá)都有顯著的改變,而Pfkfbl的表達(dá)則沒有受到明顯的影響。已有研究證明PPARy是ERK的下游分子,當(dāng)ERK通路激活后,PPARy發(fā)生磷酸化,轉(zhuǎn)錄活性降低從而抑制了分化,而Wang等的研究又證明了Brd2可抑制(?)PPARγ的轉(zhuǎn)錄活性,由此我們首先推斷Brd2可能通過ERK信號(hào)通路影響脂肪細(xì)胞的分化；其次,Glut4表達(dá)的變化直接關(guān)系到細(xì)胞的糖攝取量,而在脂肪細(xì)胞中,與糖代謝密切相關(guān)的Insulin信號(hào)通路和AMPK信號(hào)通路都可刺激GLUT4的轉(zhuǎn)位,促進(jìn)細(xì)胞對(duì)葡萄糖的攝取,其中Insulin信號(hào)是通過IGF-1R激活了IRS-1和IRS-2再進(jìn)一步激活PI3K/Akt信號(hào)通路,最后作用于GLUT4;而在AMPK信號(hào)通路中,AICAR則可不依賴于Insulin作用而直接激活A(yù)MPK,進(jìn)而刺激GLUT4的轉(zhuǎn)位。在Wang等的研究中,Brd2缺失的小鼠進(jìn)食量劇增,但其血糖水平并未隨攝糖量的急劇增加而升高,反而與野生型小鼠相比有所下降,因此,我們認(rèn)為Brd2可能通過Insulin和AMPK信號(hào)通路來共同調(diào)節(jié)脂肪細(xì)胞的糖代謝。 為了證實(shí)Brd2是否通過ERK信號(hào)通路影響脂肪細(xì)胞的分化,我們先利用RNA干擾技術(shù)使3T3-L1細(xì)胞內(nèi)的Brd2基因沉默,然后再通過轉(zhuǎn)染pmBrd2使細(xì)胞內(nèi)的Brd2過表達(dá),隨后檢測(cè)Brd2表達(dá)的變化對(duì)ERK、Raf和JNK的磷酸化水平的影響,結(jié)果表明ERK的磷酸化水平有明顯的變化,但其上游的Raf則未受到明顯的影響,同時(shí)JNK信號(hào)通路也與此過程無關(guān)；然后為了進(jìn)一步確定ERK信號(hào)通路在該過程中的作用,我們觀察了ERK激酶MEK1的特異性化學(xué)抑制劑U0126對(duì)此過程的影響,結(jié)果表明U0126可使Brd2過表達(dá)的細(xì)胞部分恢復(fù)分化,同時(shí)aP2的表達(dá)也得以回調(diào)。綜合以上結(jié)果可證實(shí)：ERK信號(hào)通路參與了Brd2調(diào)節(jié)3T3-L1前脂肪細(xì)胞向脂肪細(xì)胞分化的過程,而JNK信號(hào)通路則與之無關(guān)。 同樣,為了證實(shí)Brd2可能通過Insulin和AMPK信號(hào)通路來共同調(diào)節(jié)脂肪細(xì)胞糖代謝,我們利用RNA干擾手段下調(diào)Brd2的表達(dá)后發(fā)現(xiàn)Akt的磷酸化水平明顯升高,而通過P13K的特異性化學(xué)抑制劑LY294002可使Akt升高的活性降低,相反的,當(dāng)Brd2過表達(dá)時(shí),Akt的活性則受到了抑制；同時(shí),在研究AMPK信號(hào)通路中,我們發(fā)現(xiàn)Brd2具有負(fù)調(diào)節(jié)AMPK活性的作用：當(dāng)Brd2表達(dá)下調(diào)時(shí),AMPK的磷酸化水平明顯升高,而通過AMPK的特異性化學(xué)抑制齊(?)Compound C和Insulin都可降低AMPK的升高的活性,反之,當(dāng)Brd2過表達(dá)時(shí),AMPK的活性則受到了抑制；并且當(dāng)Brd2基因沉默時(shí),P13K的特異性化學(xué)抑制劑LY294002和AMPK的特異性化學(xué)抑制(?) Compound C都能使GLUT4蛋白表達(dá)水平顯著下降。但是Brd2表達(dá)的變化對(duì)于GSK-3的活性卻并不產(chǎn)生影響。由此,上述結(jié)果證實(shí)了我們的猜想：Brd2通過Insulin信號(hào)通路和AMPK信號(hào)通路調(diào)節(jié)3T3-L1前脂肪細(xì)胞GLUT4的表達(dá),進(jìn)而調(diào)節(jié)細(xì)胞對(duì)葡萄糖的攝取,但并不影響細(xì)胞的糖原合成。 綜上所述,在本研究中,我們證實(shí)了Brd2在3T3-L1前脂肪細(xì)胞中參與了ERK、Akt和AMPK信號(hào)通路,調(diào)控了某些相關(guān)基因的表達(dá),影響了前脂肪細(xì)胞向脂肪細(xì)胞的分化和糖代謝,但對(duì)ERK、Akt和AMPK信號(hào)通路中涉及的有關(guān)轉(zhuǎn)錄、蛋白質(zhì)合成及脂代謝還需進(jìn)一步的研究,最終更為詳細(xì)地證實(shí)Brd2在脂肪細(xì)胞分化中的作用機(jī)制。
[Abstract]:Adipose tissue is mainly composed of adipocytes. Excessive calorie intake and storage in white adipocytes in the form of triglycerides are key to obesity. Proliferation and differentiation of preadipocytes are the core of the formation of mature adipocytes. Differentiation from preadipocytes into mature adipocytes is a complex process, especially in the body. Many cytokines and hormones may regulate the development of adipocytes.
BRD2 belongs to the BET protein family. It is widely expressed in mammalian cells and has a variety of biological functions. Recent studies have shown that Brd2 gene mutation can lead to obesity in mice without diabetes, but the molecular mechanism of its role is not clear. In this study, we observed the changes of Brd2 expression in preadipocytes to fat fineness. The effect of Brd2 signaling pathway on the differentiation of preadipocytes into adipocytes was studied, which made up for the blank of this part of research.
In our study, we found that the down-regulation of Brd2 expression could induce 3T3-L1 preadipocytes to differentiate into adipocytes, even in the absence of any inducer, some cells could differentiate into adipocytes; on the contrary, when Brd2 was overexpressed, cell differentiation was almost completely inhibited even on the 8th day of induction. It is concluded that Brd2 plays an important role in the differentiation of preadipocytes into adipocytes, and we believe that the above phenomena may be due to changes in Brd2 expression that affect the expression of some differentiation-related genes or even key genes.
In order to study the effect of Brd2 expression on adipocyte differentiation-related gene expression, on the one hand, we use RNA interference technology to down-regulate the expression of Brd2 in 3T3-L1 preadipocytes; on the other hand, we use pmBrd2 vector to transfer Brd2 gene into 3T3-L1 preadipocytes, so that Brd2 overexpression in cells, and then through RT-PCR technology. The sequential expression of PPARy and C/EBPa was detected. The results showed that Brd2 had significant negative regulation on the two key genes of adipocyte differentiation, PPARy and C/EBPa. Then we detected the expression of other related genes by Q-PCR. The results showed that the expression of 422/aP2, Glut4, Leptin and Irs-1 had significant changes, while the expression of Pfkfbl was significantly different. Previous studies have shown that PPARy is the downstream molecule of ERK. When the ERK pathway is activated, PPARy phosphorylates and its transcriptional activity decreases, thus inhibiting differentiation. Wang et al. have also demonstrated that Brd2 can inhibit the transcriptional activity of (?) PPARgamma. Therefore, we first infer that Brd2 may affect the transcriptional activity of (?) PPARgamma through ERK signaling pathway. Secondly, the change of Glut4 expression is directly related to glucose uptake in adipocytes. In adipocytes, both Insulin signaling pathway and AMPK signaling pathway, which are closely related to glucose metabolism, can stimulate GLUT4 translocation and promote glucose uptake. Insulin signaling activates IRS-1 and IRS-2 reestablishment through IGF-1R. In the AMPK signaling pathway, AICAR directly activated AMPK and stimulated the translocation of GLUT4 without the effect of Insulin. In Wang et al., Brd2-deficient mice had a dramatic increase in food intake, but their blood glucose levels did not increase with the rapid increase of glucose intake, but with wild mice. Brd2 may regulate glucose metabolism in adipocytes through both Insulin and AMPK signaling pathways.
In order to confirm whether Brd2 affects adipocyte differentiation via ERK signaling pathway, we first silenced Brd2 gene in 3T3-L1 cells by RNA interference, then overexpressed Brd2 in 3T3-L1 cells by transfection of pmBrd2, and then detected the effect of Brd2 expression on the phosphorylation levels of ERK, Raf and JNK. The results showed that ERK phosphorylated. There was a significant change in the level of ERK, but the upstream Raf was not significantly affected, and the JNK signaling pathway was not related to this process; then in order to further determine the role of ERK signaling pathway in this process, we observed the effect of the specific inhibitor of ERK kinase MEK1, U0126, on this process. The results showed that U0126 could cause Brd2 to pass through the surface. These results suggest that ERK signaling pathway is involved in Brd2 regulating the differentiation of 3T3-L1 preadipocytes into adipocytes, whereas JNK signaling pathway is not involved.
Similarly, in order to confirm that Brd2 may co-regulate glucose metabolism in adipocytes through the Insulin and AMPK signaling pathways, we found that the phosphorylation level of Akt increased significantly by RNA interference, while the activity of Akt increased decreased by the specific inhibitor LY294002 of P13K, on the contrary, when Brd2 was overexpressed, Akt increased. At the same time, in the study of AMPK signaling pathway, we found that Brd2 negatively regulates the activity of AMPK: when Brd2 expression is down-regulated, AMPK phosphorylation level increases significantly, and through the specific chemical inhibition of AMPK, both Compound C and Insulin can reduce the activity of AMPK, on the contrary, when Brd2 overexpression. When Brd2 gene was silenced, both LY294002, a specific inhibitor of P13K, and Compound C, a specific inhibitor of AMPK, significantly decreased the expression of GLUT4 protein. However, the change of Brd2 expression did not affect the activity of GSK-3. Our hypothesis is that Brd2 regulates the expression of GLUT4 in 3T3-L1 preadipocytes via the Insulin and AMPK signaling pathways, thereby regulating glucose uptake, but not glycogen synthesis.
In summary, in this study, we confirmed that Brd2 participates in the ERK, Akt and AMPK signaling pathways in 3T3-L1 preadipocytes, regulates the expression of some related genes, affects the differentiation of preadipocytes into adipocytes and glucose metabolism, but the transcription, protein synthesis and lipid metabolism involved in the ERK, Akt and AMPK signaling pathways are still needed. Further studies will ultimately confirm the role of Brd2 in adipocyte differentiation.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2

【共引文獻(xiàn)】

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1 趙晶蕾;江凌勇;房兵;;叉頭框蛋白O1在骨改建中的作用[J];國際口腔醫(yī)學(xué)雜志;2014年02期

2 楊璐;魏守海;;鵝FoxO1基因cDNA序列的克隆及組織表達(dá)[J];中國畜牧獸醫(yī);2014年08期

3 李夏雨;沈守榮;武明花;李小玲;熊煒;盧建紅;周鳴;馬健;向娟娟;曾朝陽;向波;周艷宏;肖嵐;周厚德;范松青;李桂源;;多基因遺傳性腫瘤不同階段轉(zhuǎn)錄組學(xué)調(diào)控規(guī)律及其分子機(jī)制[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年07期

4 周鳴;郭馳;李夏雨;何佳瑾;徐曉杰;王賀冉;唐珂;曹利;李小玲;李桂源;;BRD7核輸出信號(hào)序列的定位與功能鑒定[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年07期

5 朱允和;周海寧;楊波;張永恒;;FoxO1基因研究進(jìn)展[J];中華實(shí)用診斷與治療雜志;2014年05期

6 黃琛;武明花;李桂源;;鼻咽癌轉(zhuǎn)錄組學(xué)研究的現(xiàn)狀與進(jìn)展[J];生物化學(xué)與生物物理進(jìn)展;2007年11期

7 李軍林;張美杰;高曉彩;;染色質(zhì)乙酰化相關(guān)BRD蛋白家族[J];生命的化學(xué);2012年02期

8 王玉霞;趙陽;鄧霖;遲希明;索琳娜;紀(jì)紅梅;;遼寧地區(qū)漢族人群中FoxO1基因rs17446614多態(tài)性與2型糖尿病的相關(guān)性[J];中國生物制品學(xué)雜志;2014年08期

9 辛雪;蘇琳;靳燁;;肌纖維及其相關(guān)基因?qū)θ馄焚|(zhì)的影響[J];食品工業(yè);2014年09期

10 李桂源;周鳴;劉華英;;鼻咽癌遺傳學(xué)研究進(jìn)展[J];中國腫瘤;2006年12期

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1 孫紅賓;殘留的偶極耦合在生物大分子溶液結(jié)構(gòu)研究中的應(yīng)用及人的MICAL-1蛋白的Calponin Homolgy結(jié)構(gòu)域和BRD7蛋白的Bromodomain結(jié)構(gòu)域的結(jié)構(gòu)與功能研究[D];中國科學(xué)技術(shù)大學(xué);2007年

2 劉華英;鼻咽癌抑瘤基因BRD7的轉(zhuǎn)錄調(diào)控研究[D];中南大學(xué);2007年

3 黃鴻達(dá);組蛋白分子伴侶Rtt106以及人的Brd2蛋白結(jié)構(gòu)與生物學(xué)功能研究[D];中國科學(xué)技術(shù)大學(xué);2010年

4 林凱東;BRD7蛋白的抗體制備和其在結(jié)直腸癌中的表達(dá)及臨床意義[D];中南大學(xué);2010年

5 楊曉紅;miR-705對(duì)絕經(jīng)后骨質(zhì)疏松小鼠的骨髓間充質(zhì)干細(xì)胞分化功能異常的調(diào)控作用[D];第四軍醫(yī)大學(xué);2013年

6 宋丹丹;蛋白酪氨酸磷酸酶1B抑制成脂并介導(dǎo)TNFα在肥胖中的作用[D];第二軍醫(yī)大學(xué);2013年

7 沈?qū)?早期應(yīng)激蛋白Egr-1及其相關(guān)信號(hào)通路調(diào)控機(jī)體血糖穩(wěn)態(tài)的研究[D];南京大學(xué);2013年

8 孫文星;二花臉豬皮下與肌內(nèi)脂肪組織基因表達(dá)譜比較及脂肪差異沉積調(diào)控機(jī)制初探[D];南京農(nóng)業(yè)大學(xué);2013年

9 江書忠;豬脂肪沉積關(guān)鍵基因的篩選及鋅指蛋白KLF13的功能研究[D];華中農(nóng)業(yè)大學(xué);2014年

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6 孫雨佳;秦川牛FoxO1基因遺傳變異及組織表達(dá)譜分析[D];西北農(nóng)林科技大學(xué);2013年

7 滕云;小鼠排卵前卵泡閉鎖過程中FSH對(duì)FOXO1基因的表達(dá)調(diào)控[D];南京農(nóng)業(yè)大學(xué);2013年

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