【摘要】：類胡蘿卜素是一類由微生物以及植物合成的天然色素,是以多個形成共軛的碳碳雙鍵為生色系結(jié)構(gòu)的脂環(huán)族或著脂肪族類型的脂溶性色素。類胡蘿卜素的種類多、涵蓋范圍廣,顏色變化是由黃色到橙紅色各不相同。普遍存在于水果和蔬菜中的類胡蘿卜素賦予各種水果和蔬菜以鮮艷明亮的顏色,同時也作為水果和蔬菜中的重要營養(yǎng)成分而備受廣泛關(guān)注。眾所周知,類胡蘿卜素是一種非常優(yōu)秀的生物類抗氧化劑。大量研究結(jié)果表明,類胡蘿卜素能夠降低一些慢性疾病的發(fā)病危險,比如冠狀動脈血管疾病和癌癥等。肥胖是脂肪細(xì)胞數(shù)量的增加以及細(xì)胞本身體積的變大而導(dǎo)致的脂肪組織過度積累的結(jié)果。存儲脂質(zhì)、分泌各種脂肪因子都是脂肪細(xì)胞的特有功能,除此之外,脂肪細(xì)胞還可以影響和調(diào)控脂肪細(xì)胞或者其它組織甚至整個生命體的生理代謝過程。肥胖病癥可以通過抑制脂肪細(xì)胞的增殖和分化來預(yù)防和治療。一些動物和細(xì)胞實(shí)驗(yàn)表明一些類胡蘿卜素,如新黃質(zhì)、巖藻黃質(zhì)以及?-隱黃質(zhì),能夠有效地抑制脂肪細(xì)胞的增殖和分化,導(dǎo)致脂肪組織發(fā)生改變。前脂肪細(xì)胞的增殖以及分化這兩個過程構(gòu)成了脂肪的合成過程,這是一個由多種基因參與的嚴(yán)密并且復(fù)雜的過程。到現(xiàn)在為止,已經(jīng)有超過600個基因被發(fā)現(xiàn)與肥胖有關(guān),其中很多研究結(jié)果顯示,過氧化物酶體增殖物激活受體gamma(PPARγ)是脂肪細(xì)胞分化的關(guān)鍵調(diào)節(jié)者之一,并參與了很多生理與病理過程,如脂質(zhì)和糖代謝過程以及糖尿病、高血壓等疾病的發(fā)生。瘦素(lep)、脂肪細(xì)胞型脂肪酸結(jié)合蛋白(FABP4)以及乙酰輔酶A羧化酶(ACACA)等蛋白都是脂肪細(xì)胞特有的并參與細(xì)胞脂肪酸代謝過程的重要蛋白�，F(xiàn)有的一些研究結(jié)果表明,脂肪細(xì)胞增殖和分化受抑制與基因PPARγ的表達(dá)量降低有關(guān);也與三種脂肪細(xì)胞特有蛋白lep、FABP4和ACACA的表達(dá)調(diào)控關(guān)聯(lián)。研究類胡蘿卜素在3T3-L1前脂肪細(xì)胞增殖和分化過程中所產(chǎn)生的影響,檢測在此過程中對基因PPARγ、lep、FABP4及ACACA蛋白的表達(dá)調(diào)控作用,探究其中的關(guān)聯(lián),有助于通過營養(yǎng)干預(yù)防止肥胖發(fā)生的醫(yī)學(xué)策略的制定與實(shí)施。本實(shí)驗(yàn)以小鼠前脂肪細(xì)胞3T3-L1為靶細(xì)胞,研究不同濃度的胭脂樹橙、番茄紅素和β-胡蘿卜素對其增殖和分化的影響,并進(jìn)一步探索該過程中PPARγ、lep、FABP4以及ACACA所發(fā)揮的可能作用。研究方法如下:(1)用DMEM細(xì)胞培養(yǎng)液(含10%FBS)在37°C、5%CO2的條件下培養(yǎng)小鼠前脂肪細(xì)胞3T3-L1,同時建立該細(xì)胞的分化模型;(2)取處于對數(shù)生長期的3T3-L1前脂肪細(xì)胞加藥處理三天,用四甲基偶氮唑鹽(MTT)法檢測細(xì)胞的存活率;(3)用經(jīng)典“雞尾酒法”進(jìn)行誘導(dǎo)分化,并在誘導(dǎo)第三天加藥處理;(4)先用油紅O染液將處理組以及對照組的脂肪細(xì)胞進(jìn)行染色,然后再應(yīng)用染色比色法來檢測脂肪細(xì)胞的分化程度;(5)利用Western Blotting技術(shù)從蛋白質(zhì)水平檢測PPARγ、lep、FABP4以及ACACA在類胡蘿卜素影響下的基因的表達(dá)的情況。研究結(jié)果顯示:1、胭脂樹橙、番茄紅素和β-胡蘿卜素對3T3-L1前脂肪細(xì)胞的增殖起抑制作用,并且與其濃度呈現(xiàn)劑量-效應(yīng)關(guān)系。2、胭脂樹橙、番茄紅素和β-胡蘿卜素對3T3-L1前脂肪細(xì)胞的分化起抑制作用。3、胭脂樹橙、番茄紅素和β-胡蘿卜素能夠下調(diào)基因PPARγ、lep和FABP4的表達(dá),同時上調(diào)基因ACACA的表達(dá)。4、以上結(jié)果表明,胭脂樹橙、番茄紅素和β-胡蘿卜素對3T3-L1前脂肪細(xì)胞的增殖和分化產(chǎn)生的抑制作用可以通過PPARγ信號通路來實(shí)現(xiàn),通過調(diào)節(jié)lep、FABP4和ACACA的蛋白表達(dá),影響細(xì)胞脂肪酸的合成與積累過程,影響細(xì)胞功能的發(fā)揮。5、研究結(jié)果可為通過功能性食品調(diào)節(jié)脂肪細(xì)胞增殖、分化及代謝,達(dá)到防治肥胖的效果提供依據(jù)與借鑒。
[Abstract]:Carotenoids are a class of natural pigments synthesized by microorganisms and plants. They are alicyclic or aliphatic pigments with conjugated carbon-carbon double bonds as chromogenic structure. Carotenoids have a wide range of types and cover a wide range of colors ranging from yellow to orange-red. They are ubiquitous in fruits and vegetables. Carotenoids in vegetables give a variety of fruits and vegetables a bright color, but also as an important nutrient in fruits and vegetables has attracted widespread attention. It is well known that carotenoids are a very good biological antioxidant. A large number of studies have shown that carotenoids can reduce some chronic diseases. Obesity is the result of excessive accumulation of adipose tissue as a result of an increase in the number of adipocytes and an increase in the size of the cells themselves. Storage of lipids and secretion of various adipocyte factors are unique functions of adipocytes. In addition, adipocytes can influence and regulate fat. Obesity can be prevented and treated by inhibiting the proliferation and differentiation of adipocytes. Some animal and cell experiments have shown that some carotenoids, such as neoxanthin, fucoxanthin and?-cryptoxanthin, can effectively inhibit the proliferation and differentiation of adipocytes. The proliferation and differentiation of preadipocytes constitute a rigorous and complex process involving a variety of genes. Up to now, more than 600 genes have been found to be associated with obesity, and many studies have shown that peroxisome proliferation is involved. Bioactivator receptor gamma (PPAR gamma) is one of the key regulators of adipocyte differentiation, and participates in many physiological and pathological processes, such as lipid and glucose metabolism, diabetes, hypertension and other diseases. Leptin (lep), fatty acid binding protein (FABP4) and acetyl coenzyme A carboxylase (ACACA) are fine lipids. Some studies have shown that the inhibition of adipocyte proliferation and differentiation is related to the decrease of PPARgamma expression, and is also related to the regulation of three adipocyte-specific proteins lep, FABP4 and ACACA expression. To investigate the effects of PPAR-gamma, lep, FABP4 and ACACA on the expression and regulation of PPAR-gamma, lep, FABP4 and ACACA proteins in the process of reproduction and differentiation, and to explore the relationship between them, it is helpful for the formulation and implementation of medical strategies to prevent obesity through nutritional intervention. The effects of adipose orange, lycopene and beta-carotene on the proliferation and differentiation of adipocytes were investigated. The possible roles of PPAR-gamma, lep, FABP4 and ACACA in this process were further explored. The main methods were as follows: (1) DMEM cell culture medium (containing 10% FBS) was used to culture mouse preadipocytes 3T3-L1 at 37 degrees C and 5% CO2, and the differentiation of the cells was established. Model; (2) 3T3-L1 preadipocytes at logarithmic growth stage were treated for three days, and the cell viability was measured by MTT method; (3) differentiation was induced by classical "cocktail method" and the treatment was added on the third day; (4) adipocytes in the treatment group and the control group were stained with oil red O staining solution. The expression of PPAR-gamma, lep, FABP4 and ACACA genes at protein level was detected by Western Blotting technique. The results showed that: 1. Proliferation of 3T3-L1 preadipocytes was affected by carotenoids, carmine orange, lycopene and beta-carotene. 2. Carmine orange, lycopene and beta-carotene inhibited the differentiation of 3T3-L1 preadipocytes. 3. Carmine orange, lycopene and beta-carotene could down-regulate the expression of PPARgamma, LEP and FABP 4, and up-regulate the expression of ACACA. Inhibitory effects of carmine, lycopene and beta-carotene on the proliferation and differentiation of 3T3-L1 preadipocytes can be achieved by PPAR-gamma signaling pathway. By regulating the protein expression of lep, FABP4 and ACACA, the synthesis and accumulation of fatty acids in cells can be affected and the function of cells can be affected. 5 Sex food can regulate the proliferation, differentiation and metabolism of adipocytes, so as to provide evidence for the prevention and treatment of obesity.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R151

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