Brd2在3T3-L1前脂肪细胞向脂肪细胞分化过程中的作用及机制研究
发布时间：2018-08-12 16:27
【摘要】：脂肪组织主要由脂肪细胞所组成,过多的热量摄入并以甘油三酯的形式储存在白色脂肪细胞内是肥胖发生的关键。前脂肪细胞的增殖分化是成熟脂肪细胞形成的核心,从前脂肪细胞分化成成熟的脂肪细胞的过程十分复杂,特别是机体内多种细胞因子和激素在脂肪细胞发生过程中可能都有调节作用。 BRD2属于BET蛋白家族,在哺乳动物细胞中广泛表达,具有多种生物学功能,最新的研究表明Brd2基因突变能导致小鼠过度肥胖而不引起糖尿病,但是其作用的分子机制并不明确。在本课题中,我们观察了Brd2表达变化对前脂肪细胞向脂肪细胞分化过程的影响,并且重点研究了Brd2参与的信号通路在影响前脂肪细胞向脂肪细胞分化作用的分子机制,弥补了这一部分研究的空白。 在研究中,我们发现Brd2表达的下调,能促使3T3-L1前脂肪细胞向脂肪细胞的分化,甚至在无任何诱导分化剂的情况下,部分细胞也能分化成脂肪细胞；相反的,当Brd2过表达时,即使诱导分化至第8天,细胞分化也几乎完全被抑制。这就更加表明Brd2在前脂肪细胞向脂肪细胞分化过程中有重要的作用,同时我们认为,上述现象的产生可能是由于Brd2表达的变化影响了一些分化相关基因甚至是关键基因的表达。 为了研究Brd2表达的变化对脂肪细胞分化相关基因表达的影响,一方面我们利用RNA干扰技术,下调3T3-L1前脂肪细胞内Brd2的表达水平；另一方面运用将pmBrd2载体将Brd2基因导入3T3-L1前脂肪细胞,使细胞内的Brd2过表达,然后先通过RT-PCR技术检测了PPARy和C/EBPa的时序性表达,结果表明Brd2对脂肪细胞分化的两个关键基因PPARy和C/EBPa都有显著的负调节作用；继而运用Q-PCR技术,我们又检测了其它相关基因的表达,结果显示,422/aP2、Glut4、 Leptin和Irs-1的表达都有显著的改变,而Pfkfbl的表达则没有受到明显的影响。已有研究证明PPARy是ERK的下游分子,当ERK通路激活后,PPARy发生磷酸化,转录活性降低从而抑制了分化,而Wang等的研究又证明了Brd2可抑制(?)PPARγ的转录活性,由此我们首先推断Brd2可能通过ERK信号通路影响脂肪细胞的分化；其次,Glut4表达的变化直接关系到细胞的糖摄取量,而在脂肪细胞中,与糖代谢密切相关的Insulin信号通路和AMPK信号通路都可刺激GLUT4的转位,促进细胞对葡萄糖的摄取,其中Insulin信号是通过IGF-1R激活了IRS-1和IRS-2再进一步激活PI3K/Akt信号通路,最后作用于GLUT4;而在AMPK信号通路中,AICAR则可不依赖于Insulin作用而直接激活AMPK,进而刺激GLUT4的转位。在Wang等的研究中,Brd2缺失的小鼠进食量剧增,但其血糖水平并未随摄糖量的急剧增加而升高,反而与野生型小鼠相比有所下降,因此,我们认为Brd2可能通过Insulin和AMPK信号通路来共同调节脂肪细胞的糖代谢。 为了证实Brd2是否通过ERK信号通路影响脂肪细胞的分化,我们先利用RNA干扰技术使3T3-L1细胞内的Brd2基因沉默,然后再通过转染pmBrd2使细胞内的Brd2过表达,随后检测Brd2表达的变化对ERK、Raf和JNK的磷酸化水平的影响,结果表明ERK的磷酸化水平有明显的变化,但其上游的Raf则未受到明显的影响,同时JNK信号通路也与此过程无关；然后为了进一步确定ERK信号通路在该过程中的作用,我们观察了ERK激酶MEK1的特异性化学抑制剂U0126对此过程的影响,结果表明U0126可使Brd2过表达的细胞部分恢复分化,同时aP2的表达也得以回调。综合以上结果可证实：ERK信号通路参与了Brd2调节3T3-L1前脂肪细胞向脂肪细胞分化的过程,而JNK信号通路则与之无关。 同样,为了证实Brd2可能通过Insulin和AMPK信号通路来共同调节脂肪细胞糖代谢,我们利用RNA干扰手段下调Brd2的表达后发现Akt的磷酸化水平明显升高,而通过P13K的特异性化学抑制剂LY294002可使Akt升高的活性降低,相反的,当Brd2过表达时,Akt的活性则受到了抑制；同时,在研究AMPK信号通路中,我们发现Brd2具有负调节AMPK活性的作用：当Brd2表达下调时,AMPK的磷酸化水平明显升高,而通过AMPK的特异性化学抑制齐(?)Compound C和Insulin都可降低AMPK的升高的活性,反之,当Brd2过表达时,AMPK的活性则受到了抑制；并且当Brd2基因沉默时,P13K的特异性化学抑制剂LY294002和AMPK的特异性化学抑制(?) Compound C都能使GLUT4蛋白表达水平显著下降。但是Brd2表达的变化对于GSK-3的活性却并不产生影响。由此,上述结果证实了我们的猜想：Brd2通过Insulin信号通路和AMPK信号通路调节3T3-L1前脂肪细胞GLUT4的表达,进而调节细胞对葡萄糖的摄取,但并不影响细胞的糖原合成。 综上所述,在本研究中,我们证实了Brd2在3T3-L1前脂肪细胞中参与了ERK、Akt和AMPK信号通路,调控了某些相关基因的表达,影响了前脂肪细胞向脂肪细胞的分化和糖代谢,但对ERK、Akt和AMPK信号通路中涉及的有关转录、蛋白质合成及脂代谢还需进一步的研究,最终更为详细地证实Brd2在脂肪细胞分化中的作用机制。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329.2
本文编号：2179613
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329.2
【共引文献】
相关期刊论文 前10条
1 赵晶蕾;江凌勇;房兵;;叉头框蛋白O1在骨改建中的作用[J];国际口腔医学杂志;2014年02期
2 杨璐;魏守海;;鹅FoxO1基因cDNA序列的克隆及组织表达[J];中国畜牧兽医;2014年08期
3 李夏雨;沈守荣;武明花;李小玲;熊炜;卢建红;周鸣;马健;向娟娟;曾朝阳;向波;周艳宏;肖岚;周厚德;范松青;李桂源;;多基因遗传性肿瘤不同阶段转录组学调控规律及其分子机制[J];中南大学学报(医学版);2011年07期
4 周鸣;郭驰;李夏雨;何佳瑾;徐晓杰;王贺冉;唐珂;曹利;李小玲;李桂源;;BRD7核输出信号序列的定位与功能鉴定[J];中南大学学报(医学版);2011年07期
5 朱允和;周海宁;杨波;张永恒;;FoxO1基因研究进展[J];中华实用诊断与治疗杂志;2014年05期
6 黄琛;武明花;李桂源;;鼻咽癌转录组学研究的现状与进展[J];生物化学与生物物理进展;2007年11期
7 李军林;张美杰;高晓彩;;染色质乙酰化相关BRD蛋白家族[J];生命的化学;2012年02期
8 王玉霞;赵阳;邓霖;迟希明;索琳娜;纪红梅;;辽宁地区汉族人群中FoxO1基因rs17446614多态性与2型糖尿病的相关性[J];中国生物制品学杂志;2014年08期
9 辛雪;苏琳;靳烨;;肌纤维及其相关基因对肉品质的影响[J];食品工业;2014年09期
10 李桂源;周鸣;刘华英;;鼻咽癌遗传学研究进展[J];中国肿瘤;2006年12期
相关博士学位论文 前10条
1 孙红宾;残留的偶极耦合在生物大分子溶液结构研究中的应用及人的MICAL-1蛋白的Calponin Homolgy结构域和BRD7蛋白的Bromodomain结构域的结构与功能研究[D];中国科学技术大学;2007年
2 刘华英;鼻咽癌抑瘤基因BRD7的转录调控研究[D];中南大学;2007年
3 黄鸿达;组蛋白分子伴侣Rtt106以及人的Brd2蛋白结构与生物学功能研究[D];中国科学技术大学;2010年
4 林凯东;BRD7蛋白的抗体制备和其在结直肠癌中的表达及临床意义[D];中南大学;2010年
5 杨晓红;miR-705对绝经后骨质疏松小鼠的骨髓间充质干细胞分化功能异常的调控作用[D];第四军医大学;2013年
6 宋丹丹;蛋白酪氨酸磷酸酶1B抑制成脂并介导TNFα在肥胖中的作用[D];第二军医大学;2013年
7 沈宁;早期应激蛋白Egr-1及其相关信号通路调控机体血糖稳态的研究[D];南京大学;2013年
8 孙文星;二花脸猪皮下与肌内脂肪组织基因表达谱比较及脂肪差异沉积调控机制初探[D];南京农业大学;2013年
9 江书忠;猪脂肪沉积关键基因的筛选及锌指蛋白KLF13的功能研究[D];华中农业大学;2014年
10 杨莘;PI3K/Akt信号通路与天然免疫限制性因子SAMHD1在PRRSV感染过程中的作用研究[D];中国农业科学院;2014年
相关硕士学位论文 前8条
1 宋佳巍;营养水平对不同品种鹅生长发育及MyoG、Foxo1基因表达的影响[D];吉林农业大学;2013年
2 陈小龙;衰老小鼠视网膜中相关基因的表达及山茱萸多糖的干预作用[D];佳木斯大学;2013年
3 罗玮;n-6/n-3多不饱和脂肪酸构成对3T3-L1脂肪细胞脂联素和PPARγ表达的调节研究[D];南方医科大学;2013年
4 欧阳正朝;Nodal蛋白在肺腺癌中的表达及意义[D];中南大学;2013年
5 熊燕;C/EBPβ和FoxO1通过反馈环和蛋白互作调控猪前体脂肪细胞分化[D];西北农林科技大学;2013年
6 孙雨佳;秦川牛FoxO1基因遗传变异及组织表达谱分析[D];西北农林科技大学;2013年
7 滕云;小鼠排卵前卵泡闭锁过程中FSH对FOXO1基因的表达调控[D];南京农业大学;2013年
8 李凡;磷酸化FoxO1在高糖诱导人肾小管上皮细胞脂质沉积中的作用[D];河北医科大学;2014年
，本文编号：2179613
本文链接：https://www.wllwen.com/xiyixuelunwen/2179613.html
最近更新
教材专著
