类胡萝卜素对小鼠前脂肪细胞3T3-L1增殖和分化的影响

发布时间：2018-09-08 11:57

【摘要】：类胡萝卜素是一类由微生物以及植物合成的天然色素,是以多个形成共轭的碳碳双键为生色系结构的脂环族或着脂肪族类型的脂溶性色素。类胡萝卜素的种类多、涵盖范围广,颜色变化是由黄色到橙红色各不相同。普遍存在于水果和蔬菜中的类胡萝卜素赋予各种水果和蔬菜以鲜艳明亮的颜色,同时也作为水果和蔬菜中的重要营养成分而备受广泛关注。众所周知,类胡萝卜素是一种非常优秀的生物类抗氧化剂。大量研究结果表明,类胡萝卜素能够降低一些慢性疾病的发病危险,比如冠状动脉血管疾病和癌症等。肥胖是脂肪细胞数量的增加以及细胞本身体积的变大而导致的脂肪组织过度积累的结果。存储脂质、分泌各种脂肪因子都是脂肪细胞的特有功能,除此之外,脂肪细胞还可以影响和调控脂肪细胞或者其它组织甚至整个生命体的生理代谢过程。肥胖病症可以通过抑制脂肪细胞的增殖和分化来预防和治疗。一些动物和细胞实验表明一些类胡萝卜素,如新黄质、岩藻黄质以及?-隐黄质,能够有效地抑制脂肪细胞的增殖和分化,导致脂肪组织发生改变。前脂肪细胞的增殖以及分化这两个过程构成了脂肪的合成过程,这是一个由多种基因参与的严密并且复杂的过程。到现在为止,已经有超过600个基因被发现与肥胖有关,其中很多研究结果显示,过氧化物酶体增殖物激活受体gamma(PPARγ)是脂肪细胞分化的关键调节者之一,并参与了很多生理与病理过程,如脂质和糖代谢过程以及糖尿病、高血压等疾病的发生。瘦素(lep)、脂肪细胞型脂肪酸结合蛋白(FABP4)以及乙酰辅酶A羧化酶(ACACA)等蛋白都是脂肪细胞特有的并参与细胞脂肪酸代谢过程的重要蛋白。现有的一些研究结果表明,脂肪细胞增殖和分化受抑制与基因PPARγ的表达量降低有关;也与三种脂肪细胞特有蛋白lep、FABP4和ACACA的表达调控关联。研究类胡萝卜素在3T3-L1前脂肪细胞增殖和分化过程中所产生的影响,检测在此过程中对基因PPARγ、lep、FABP4及ACACA蛋白的表达调控作用,探究其中的关联,有助于通过营养干预防止肥胖发生的医学策略的制定与实施。本实验以小鼠前脂肪细胞3T3-L1为靶细胞,研究不同浓度的胭脂树橙、番茄红素和β-胡萝卜素对其增殖和分化的影响,并进一步探索该过程中PPARγ、lep、FABP4以及ACACA所发挥的可能作用。研究方法如下:(1)用DMEM细胞培养液(含10%FBS)在37°C、5%CO2的条件下培养小鼠前脂肪细胞3T3-L1,同时建立该细胞的分化模型;(2)取处于对数生长期的3T3-L1前脂肪细胞加药处理三天,用四甲基偶氮唑盐(MTT)法检测细胞的存活率;(3)用经典“鸡尾酒法”进行诱导分化,并在诱导第三天加药处理;(4)先用油红O染液将处理组以及对照组的脂肪细胞进行染色,然后再应用染色比色法来检测脂肪细胞的分化程度;(5)利用Western Blotting技术从蛋白质水平检测PPARγ、lep、FABP4以及ACACA在类胡萝卜素影响下的基因的表达的情况。研究结果显示:1、胭脂树橙、番茄红素和β-胡萝卜素对3T3-L1前脂肪细胞的增殖起抑制作用,并且与其浓度呈现剂量-效应关系。2、胭脂树橙、番茄红素和β-胡萝卜素对3T3-L1前脂肪细胞的分化起抑制作用。3、胭脂树橙、番茄红素和β-胡萝卜素能够下调基因PPARγ、lep和FABP4的表达,同时上调基因ACACA的表达。4、以上结果表明,胭脂树橙、番茄红素和β-胡萝卜素对3T3-L1前脂肪细胞的增殖和分化产生的抑制作用可以通过PPARγ信号通路来实现,通过调节lep、FABP4和ACACA的蛋白表达,影响细胞脂肪酸的合成与积累过程,影响细胞功能的发挥。5、研究结果可为通过功能性食品调节脂肪细胞增殖、分化及代谢,达到防治肥胖的效果提供依据与借鉴。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R151

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