【摘要】：機(jī)體內(nèi)脂肪主要有兩類(lèi):與脂肪堆積有關(guān)的白色脂肪組織(White adipose tissue,WAT)和脂肪代謝有關(guān)的棕色脂肪組織(Brown adipose tissue,BAT)。前者通過(guò)甘油三酯的形式儲(chǔ)存能量,與肥胖癥,糖尿病和血管類(lèi)疾病的發(fā)生密切相關(guān),后者則通過(guò)氧化磷酸化代謝脂肪,負(fù)責(zé)能量的提供,可有效抗肥胖。有研究證實(shí),在適當(dāng)?shù)拇碳は?白色脂肪可以被轉(zhuǎn)化為棕色脂肪,即白色脂肪棕色化。因此闡明白色脂肪棕色化的具體分子機(jī)制對(duì)于人類(lèi)的健康具有重要意義。與脂肪發(fā)生有關(guān)的因子有很多,其中血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor,VEGF-A)近年來(lái)被發(fā)現(xiàn)在脂肪組織中大量表達(dá),并且與脂肪組織發(fā)育及能量代謝密切相關(guān)。我們建立了四環(huán)素-Vegfa可逆調(diào)節(jié)小鼠模型,并成功介導(dǎo)白色脂肪棕色化。在此進(jìn)程中,除WAT和BAT關(guān)鍵調(diào)控因子Leptin,Prdm16,Ucp1,Cidea,Bmp4和Bmp7發(fā)生顯著改變之外,Gata1也發(fā)生了顯著性上調(diào)。過(guò)往對(duì)GATA1的報(bào)道主要集中于其在紅系分化中的調(diào)控作用,在脂肪分化中的研究較少,然而其是否也在脂肪分化與能量代謝中發(fā)揮了重要調(diào)節(jié)作用正是我們本研究的主要內(nèi)容。在本論文中,我們對(duì)小鼠不同脂肪組織間Gata1的表達(dá)特點(diǎn)及其在白色脂肪棕色化進(jìn)程中的作用及相關(guān)機(jī)制進(jìn)行了研究。首先我們對(duì)小鼠體內(nèi)WAT和BAT進(jìn)行了分離并分別提取RNA,Real-time PCR結(jié)果顯示Gata1在BAT中的表達(dá)量要遠(yuǎn)遠(yuǎn)高于其在WAT中的表達(dá),這與同時(shí)檢測(cè)的棕色脂肪調(diào)控因子Prdm16、Ucp1和Cidea等的表達(dá)特點(diǎn)是一致的。繼而利用構(gòu)建好的四環(huán)素-Vegfa可逆調(diào)節(jié)小鼠模型,我們發(fā)現(xiàn)在白色脂肪棕色化的進(jìn)程中Gata1發(fā)生了明顯的上調(diào),并且其表達(dá)與Vegfa呈現(xiàn)顯著性的負(fù)相關(guān)。繼而我們利用pc DNA3.1(-)載體及慢病毒載體pco EGFP-puro分別構(gòu)建了適用于瞬時(shí)轉(zhuǎn)染和慢病毒侵染的Gata1過(guò)表達(dá)質(zhì)粒,并在不同細(xì)胞系中進(jìn)行了瞬時(shí)轉(zhuǎn)染和慢病毒侵染實(shí)驗(yàn)。Real-time PCR檢測(cè)結(jié)果顯示,不論在3T3-L1細(xì)胞系還是原代脂肪前體細(xì)胞中,外源過(guò)表達(dá)Gata1后均能夠上調(diào)相同的棕色脂肪調(diào)控因子的表達(dá),如Bmp4,Pgc1-α和Cidea;但是并不影響B(tài)mp7、Prdm16和Ucp1的表達(dá)。我們的實(shí)驗(yàn)結(jié)果揭示在脂肪分化進(jìn)程中,Gata1的表達(dá)與棕色脂肪發(fā)生及白色脂肪棕色化密切相關(guān);其作用的發(fā)揮可能是通過(guò)上調(diào)Bmp4,Pgc1-α和Cidea等棕色脂肪調(diào)控因子的表達(dá)而實(shí)現(xiàn)的。GATA1在白色脂肪棕色化中的作用及其機(jī)制的闡明既補(bǔ)充了GATA1已有的生物學(xué)功能,為能量代謝研究提供了新的線(xiàn)索,同時(shí)也為預(yù)防和控制肥胖提供了一個(gè)潛在靶點(diǎn)。
[Abstract]:There are two main types of fat in the body: (White adipose tissue,WAT (white adipose tissue associated with fat accumulation) and (Brown adipose tissue,BAT (brown adipose tissue related to fat metabolism). The former is closely related to obesity, diabetes and vascular diseases by storing energy in the form of triglycerides, while the latter can effectively resist obesity by oxidizing phosphorylated metabolized fat, which is responsible for energy supply. Studies have shown that white fat can be turned into brown fat, or white fat, brown with appropriate stimulation. Therefore, it is important for human health to clarify the specific molecular mechanism of white fat browning. There are many factors related to adipogenesis, among which vascular endothelial growth factor (Vascular endothelial growth factor,VEGF-A) has been found to be highly expressed in adipose tissue in recent years, and is closely related to adipose tissue development and energy metabolism. We established a mouse model of reversible regulation of tetracycline-Vegfa and successfully mediated white fat browning. In addition to the significant changes in WAT and BAT key regulatory factors, Leptin,Prdm16,Ucp1,Cidea,Bmp4 and Bmp7, Gata1 also increased significantly during this process. Previous reports on GATA1 mainly focus on its regulatory role in erythroid differentiation, but less on adipose differentiation. However, whether GATA1 also plays an important regulatory role in adipodifferentiation and energy metabolism is the main content of this study. In this paper, we studied the expression of Gata1 in different adipose tissues of mice and its role in the brown process of white fat and its related mechanisms. First, we isolated WAT and BAT from mice and extracted RNA,Real-time PCR respectively. The results showed that the expression of Gata1 in BAT was much higher than that in WAT. This was consistent with the expression of brown fat regulatory factors Prdm16,Ucp1 and Cidea. Then we established a mouse model of reversible regulation of tetracycline-Vegfa. We found that Gata1 was significantly up-regulated in the course of brown white fat and its expression was negatively correlated with Vegfa. Then we constructed Gata1 overexpression plasmids suitable for transient transfection and lentivirus infection using pc DNA3.1 (-) vector and lentivirus vector pco EGFP-puro, and carried out transient transfection and lentivirus infection experiments in different cell lines. In both 3T3-L1 cell lines and primary adipose progenitor cells, exogenous overexpression of Gata1 could up-regulate the expression of the same brown fat regulatory factors, such as Bmp4,Pgc1- 偽 and Cidea;, but did not affect the expression of Bmp7,Prdm16 and Ucp1. Our results showed that the expression of Gata1 was closely related to brown adipogenesis and white fat browning during adipose differentiation. The role of GATA1 in the brown fat browning may be achieved by upregulating the expression of brown fat regulatory factors such as Bmp4,Pgc1- 偽 and Cidea. The role of GATA1 in the browning of white fat and its mechanism may complement the existing biological functions of GATA1. It provides a new clue for energy metabolism research and a potential target for the prevention and control of obesity.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R589.2

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