内源性n-3 PUFA调节小鼠体重过程中下丘脑食欲神经肽的变化

发布时间：2018-06-13 01:44

本文选题：n-3 + PUFA　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:近来的研究发现,n-3多不饱和脂肪酸(polyunsaturated fatty acids,PUFA)可以有效地抵抗肥胖,减少一些肥胖引发的相关疾病的发生。Fat-1基因来源于秀丽隐杆线虫,能够编码n-3 PUFA脱氢酶。该酶以n-6 PUFA为底物合成n-3 PUFA。Fat-1转基因小鼠正是携带了这种fat-1基因,可以增加内源性的n-3 PUFA含量,平衡体内n-6/n-3 PUFA的比例,所以降低了体重。但其详细的机理不详。因此,本研究利用fat-1转基因小鼠作为模型,研究下丘脑食欲神经肽和解偶联蛋白(UCP)在fat-1小鼠中的变化----对抑制体重所起的作用及可能的机理。方法:Fat-1转基因小鼠来自于美国哈佛医学院Jing-Xuan Kang教授和澳门大学Jian-Bo Wan博士赠送的SPF级小鼠。采用fat-1转基因小鼠与普通野生型C57/BL6小鼠杂交,获得同窝的雄性小鼠。采用普通的PCR进行基因型鉴定,将6周龄健康小鼠分为fat-1转基因组和非转基因组。每周测量小鼠体重和体长,持续8周。实验结束时处死两组小鼠,称量小鼠内脏周围脂肪重量;采用油红O染色,染小鼠肝脏切片中的脂肪滴;采集血液测定两组小鼠的高密度脂蛋白胆固醇(HDL-C)含量、低密度脂蛋白胆固醇(LDL-C)含量和总胆固醇(TC)含量;采用免疫荧光技术,对下丘脑食欲相关神经肽Neuropeptide Y(NPY)、Agouti-related peptides(Ag RP)、Proopiomelanocortin(POMC)、Cocaine and amphetamine regulated transcript(CART)和Ghrelin进行半定量分析;采用实时荧光定量PCR,对脂肪组织中Leptin m RNA、UCP-1 m RNA和UCP-2 m RNA,血液中Leptin m RNA进行相对定量分析;采用Western blotting,对脂肪组织中Leptin和UCP-2蛋白进行相对表达量分析。结果:每周测量体重与体长表明,fat-1转基因小鼠的体重/体长比例明显低于非转基因小鼠(P0.05);fat-1转基因小鼠的脂肪重量明显低于非转基因小鼠(P0.05);fat-1转基因小鼠肝脏中的脂肪滴明显少于非转基因小鼠;fat-1转基因小鼠血清中的HDL-C、LDL-C和TC含量明显低于非转基因小鼠(P0.05);fat-1转基因小鼠与其同窝野生型小鼠相比,体内下丘脑饮食相关基因抑食神经肽POMC、CART的相对表达量显著上调(P0.05),促食神经肽NPY和Ag RP的相对表达量显著下调(P0.05),生长肽Ghrelin的相对表达量显著上调(P0.05);实时荧光定量PCR结果显示,脂肪组织中,fat-1转基因小鼠肥胖相关基因Leptin m RNA的相对表达量显著下调(P0.05),UCP-1 m RNA和UCP-2 m RNA相对表达量显著上调(P0.05),血液中Leptin m RNA差异不明显(P0.05);Western blotting结果显示,脂肪组织中,Leptin蛋白的相对表达量差异不明显(P0.05),UCP-2蛋白相对表达量显著上调(P0.05)。结论:本实验研究结果表明,fat-1转基因小鼠体内增加的n-3 PUFA水平可以改善能量代谢,控制小鼠的体重;其机理可能是通过影响小鼠下丘脑饮食相关神经肽基因和解耦联蛋白UCP的表达,导致体重降低。Leptin的表达在fat-1转基因小鼠中并没有显著变化,说明fat-1小鼠体重的减轻不是由Leptin引起的,可能是增加UCP的表达而产生的。但n-3 PUFA调节体重详细的机理还需要进一步的研究。
[Abstract]:Objective: recent studies have found that n-3 polyunsaturated fatty acids (polyunsaturated fatty acids, PUFA) can effectively resist obesity and reduce the incidence of some associated diseases caused by obesity. The.Fat-1 gene is derived from Caenorhabditis elegans, and can encode n-3 PUFA dehydrogenase. This enzyme uses n-6 PUFA as the substrate to synthesize n-3 PUFA.Fat-1 transgenic mice. With this fat-1 gene, it can increase the endogenous n-3 PUFA content and balance the proportion of n-6/n-3 PUFA in the body, thus reducing the weight. However, the detailed mechanism is not detailed. Therefore, this study uses fat-1 transgenic mice as a model to study the changes in the hypothalamic appetite neuropeptide and the coupling protein (UCP) in fat-1 mice. The effect and possible mechanism of body weight making. Methods: Fat-1 transgenic mice were derived from Professor Jing-Xuan Kang of Harvard Medical School in the United States and SPF mice given by Dr. Jian-Bo Wan of Macao University. Using fat-1 transgenic mice and common wild C57/BL6 mice, the male mice of the same nest were obtained. Common PCR was used for genotyping. The 6 weeks old healthy mice were divided into fat-1 rotation genome and non rotation genome. The weight and body length of mice were measured weekly for 8 weeks. At the end of the experiment, two groups of mice were killed and the fat weight around the viscera in mice was weighed. The fat droplets in the liver slices were stained with oil red O, and the high density lipoprotein gallbladder of the two groups of mice was collected. The content of sterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC), and immunofluorescence technique were used to determine the hypothalamic appetite related neuropeptide Neuropeptide Y (NPY), Agouti-related peptides (Ag RP) and Proopiomelanocortin (POMC). The relative quantitative analysis of Leptin m RNA, UCP-1 m RNA and UCP-2 m RNA in the adipose tissue was carried out by real time fluorescence quantitative PCR, and the relative expression of the protein in the fat tissue was analyzed. The long proportion was significantly lower than that of non transgenic mice (P0.05); the fat weight of fat-1 transgenic mice was significantly lower than that of non transgenic mice (P0.05), and the fat drops in the liver of fat-1 transgenic mice were obviously less than those of non transgenic mice; the content of HDL-C, LDL-C and TC in the serum of fat-1 transgenic mice was significantly lower than that of non transgenic mice (P0.05); fat-1 transgenic mice were genetically modified. The relative expression of neuropeptide POMC in the hypothalamic diet related genes was significantly up (P0.05), and the relative expression of neuropeptide NPY and Ag RP was significantly down (P0.05), and the relative expression of NGP Ghrelin was significantly up (P0.05). Real time fluorescent quantitative PCR showed that the fat tissue was in fat tissue. In the fat-1 transgenic mice, the relative expression of Leptin m RNA was significantly down (P0.05), and the relative expression of UCP-1 m RNA and UCP-2 m increased significantly (P0.05). The relative expression of protein was significantly up-regulated (P0.05). Conclusion: the results of this study showed that the increase of n-3 PUFA level in fat-1 transgenic mice could improve the energy metabolism and control the weight of mice. The mechanism may be caused by the influence of the expression of neuropeptide related neuropeptide and uncoupling protein UCP in the hypothalamus diet of mice, resulting in the weight loss of.Lepti. The expression of N did not change significantly in fat-1 transgenic mice, indicating that the loss of weight in fat-1 mice was not caused by Leptin and may be produced by increasing the expression of UCP. But the mechanism of n-3 PUFA regulation of body weight needs further study.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R338

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