自由摄食状态下不同膳食脂肪供能比及产能营养素结构对大鼠肥胖相关指标的影响

发布时间：2018-07-18 14:27

【摘要】：目的:为多角度全面分析自由摄食状态下不同脂肪供能比及产能营养素结构膳食对肥胖等慢性病相关危险因素的影响,本研究通过用不同脂肪供能比及产能营养素结构饲料对大鼠进行为期10周的喂养,期间记录大鼠的进食、体重等一般情况,测量血压、血生化指标,检测脂肪因子、食欲调节激素水平及其基因表达情况,了解肠道菌群种类及相对丰度情况,分析各项指标在不同组间的差异及其与肥胖发生的关系,为进一步揭示膳食结构是否与肥胖发生相关及科学指导合理膳食提供基础参考数据。方法:1、自由摄食状态下不同膳食脂肪供能比及产能营养素结构对大鼠血液指标及相关基因表达的影响70只健康雄性SD大鼠经适应性喂养3周后,根据其体重增长情况随机分为7组,每组各10只,分别饲喂低脂高碳水组、低脂高蛋白组、普通组、中脂低碳水组、中脂低蛋白组、高脂低碳水组和高脂低蛋白组饲料共10周。大鼠自由摄食饮水,单笼饲养。每周测量大鼠体重、体长,正式实验开始第0、5、10周测量血压并进行内眦采血,检测血糖、血脂、游离脂肪酸、胰岛素、脂联素(Adiponectin)、瘦素(Leptin)、脂多糖结合蛋白(Lipopoysacchride Binding Protein,LBP)、食欲刺激素(Ghrelin)和肥胖抑制素(Obestatin)水平。实验结束时处死大鼠并取肾周脂肪、附睾周脂肪和大网膜脂肪称重,同时留取附睾周脂肪组织和胃组织,提取各组织总mRNA,反转录成cDNA进行PCR扩增,使用实时荧光定量PCR方法检测脂肪组织中脂联素、瘦素基因和胃组织中生长激素促分泌素受体(Growth Hormone Secretagogue Receptor,GHSR)、G 蛋白偶联受体 39(G Protein-coupled Receptor 39,GPR39)基因的表达情况。2、自由摄食状态下不同膳食脂肪供能比及产能营养素结构对大鼠肠道菌群结构的影响在对大鼠分组后(第0周),每组随机选取6只大鼠,无菌采集每只大鼠粪便约1.0 g,并在实验第10周末采用相同方法采集相同大鼠粪便。提取粪便样本基因组总DNA,检测DNA浓度和纯度,之后对样本DNA进行PCR扩增、纯化。将质检合格的DNA文库在Illumina Miseq平台上进行测序分析,测序读长为2×300 bp。将所得序列进行拼接、质控、过滤,比对到已知16SrDNA数据库并进行物种分类学分析、α多样性分析、β多样性分析和主成分分析等。结果:1、自由摄食状态下不同膳食脂肪供能比及产能营养素结构对大鼠血液指标及相关基因表达的影响第10周末,高脂低碳水组和高脂低蛋白组大鼠血清胰岛素和胰岛素抵抗指数均高于低脂高碳水组;中脂低蛋白组大鼠血清总胆固醇、低密度脂蛋白胆固醇水平均低于普通组;中脂低蛋白组、高脂低碳水组、高脂低蛋白组大鼠血清高密度脂蛋白胆固醇水平均低于普通组;低脂高碳水组Ghrelin/Obestatin比值低于中脂低蛋白组和高脂低蛋白组。第10周末,低脂高碳水组大鼠脂肪组织脂联素mRNA相对表达量高于中脂低碳水组,低脂高蛋白组高于普通组和中脂低碳水组;高脂低蛋白组大鼠胃组织GHSR mRNA相对表达量高于低脂高蛋白组;普通组大鼠胃组织GPR39 mRNA相对表达量低于除低脂高蛋白组的其他组,低脂高蛋白组低于高脂低碳水组。各组大鼠在各时期平均每日能量摄入量、体重、体长、Lee's指数、体脂比、血压、空腹血糖、甘油三酯、游离脂肪酸、瘦素、脂联素、Ghrelin、Obestatin水平以及脂肪组织瘦素基因表达均无统计学差异。2、自由摄食状态下不同膳食脂肪供能比及产能营养素结构对大鼠肠道菌群结构的影响第10周与第0周相比,大鼠肠道菌群出现拟杆菌门减少、厚壁菌门增加、变形菌门增加的趋势。第10周,各组之间肠道菌群丰富度和多样性没有差异。中脂低蛋白组大鼠肠道菌群中拟杆菌门相对丰度最低,厚壁菌门与低脂高蛋白组、中脂低碳水组、高脂低碳水组相比显著升高,拟杆菌门/厚壁菌门比值低于低脂高蛋白组和中脂低碳水组;低脂高碳水组大鼠肠道菌群中S24-7相对丰度高于中脂低蛋白组;低脂高蛋白组Defluviitaleaceae高于高脂低碳水组;中脂低蛋白组VadinBB60高于除高脂低蛋白组外所有组;低脂高碳水组Incertae_Sedis高于中脂低碳水组和高脂低碳水组。结论:1、不同膳食脂肪供能比和产能营养素结构对大鼠Lee's指数、体脂比、血压、血糖、甘油三酯、游离脂肪酸、瘦素、脂联素、Ghrelin、Obestatin水平以及脂肪组织瘦素基因表达等无影响。2、高脂肪供能比膳食会引起高密度脂蛋白胆固醇的降低和胰岛素抵抗的产生,可能使大鼠处于体重未改变而糖脂代谢紊乱的状态。3、高碳水化合物供能比膳食会提高脂联素基因的表达,具体机制仍需进一步研究。4、自由摄食状态下,不同脂肪供能比和产能营养素结构饲料对大鼠肠道菌群丰富度和多样性没有影响。5、与高脂肪膳食相比,高碳水化合物供能比膳食更有助于S24-7、D)efluviitaleaceae和incertae_Sedis的增殖,具体生物学意义有待于进一步研究。
[Abstract]:Objective: to analyze the effects of different fat energy supply ratio and productivity nutrient structure diet on obesity and other chronic disease risk factors in the free feeding state. This study was carried out by feeding rats with different fat energy supply ratio and productive nutrient structure feed for 10 weeks, during which the diet and weight of rats were recorded. In the case of blood pressure, blood biochemical index, fat factor, appetite regulating hormone level and gene expression, the species and relative abundance of intestinal flora were understood, the differences among different groups and the relationship with obesity were analyzed, so as to further reveal whether the dietary structure was related to obesity and scientific guidance. The basic reference data of rational diet were provided. Methods: 1, the effects of different dietary fat supply and productivity on the blood indexes and related genes expression in 70 healthy male SD rats were randomly divided into 7 groups after 3 weeks of adaptive feeding, and 10 rats in each group were fed low fat, respectively. High carbon water group, low fat and high protein group, medium fat low carbon water group, low fat low protein group, high fat low carbon water group and high fat low protein group feed for 10 weeks. Rats were free to feed drinking water, single cage feeding. Measure the body weight and body length of rats every week. The blood pressure was measured at the beginning of the 0,5,10 week, and the blood pressure was measured in the inner canthus, blood glucose, blood lipid, free fat were detected. Fatty acid, insulin, adiponectin (Adiponectin), leptin (Leptin), lipopolysaccharide binding protein (Lipopoysacchride Binding Protein, LBP), appetite stimulating hormone (Ghrelin) and obesity inhibin (Obestatin) level. At the end of the experiment, rats were killed and perinenal fat, epididymal fat and omental fat were weighed, and epididymal adipose tissue and epididymal adipose tissue were left at the end of the experiment. Gastric tissue, extract the total mRNA of each tissue, reverse transcripted into cDNA for PCR amplification, and use real-time fluorescence quantitative PCR to detect adiponectin, leptin gene and growth hormone secretocadotropin receptor (Growth Hormone Secretagogue Receptor, GHSR), G protein coupled receptor 39 (G Protein-coupled 39,) gene in the tissues of the stomach The effect of.2, the effect of different dietary fat supply and productivity on the structure of intestinal microflora in rats under free feeding state (zeroth weeks), 6 rats in each group were randomly selected, and the feces of each rat were collected by 1 g, and the feces were collected by the same method at the end of the experiment and the feces were extracted by the same method. Then the total genomic DNA of the sample was collected and the concentration and purity of DNA were detected. Then the sample DNA was amplified and purified by PCR. The qualified DNA library was sequenced and analyzed on the Illumina Miseq platform. The sequence was sequenced by 2 x 300 bp., and the sequence was spliced, the quality control and filtration were compared to the known 16SrDNA database and the taxonomic analysis of the species was carried out. The alpha was much more. Results of sample analysis, beta diversity analysis and principal component analysis. The results were as follows: 1, the effect of different dietary fat donor energy ratio and productivity nutrient structure on blood indexes and related gene expression in rats at free feeding state for tenth weeks, the serum insulin and insulin resistance index of high fat low carbon water group and high fat low protein group were higher than those of low fat. In the carbon and water group, the serum total cholesterol and low density lipoprotein cholesterol levels in the low fat low protein group were lower than those in the normal group, and the high density lipoprotein cholesterol levels in the low fat low protein group, the high fat and low carbon water group and the high fat and low protein group were lower than those in the normal group, and the Ghrelin/Obestatin ratio in the low fat and high carbon water group was lower than the middle fat low protein group and the high fat group. The relative expression of adiponectin mRNA in adipose tissue of low fat and high carbon water group was higher than that of low fat and low carbon water group at the weekend of low fat and high carbon water group, and the low fat and high protein group was higher than that of the normal group and medium fat low carbon water group. The relative expression of GHSR mRNA in the gastric tissue of the high fat and low protein group was higher than that of the low fat and high protein group, and the relative expression of GPR39 mRNA in the gastric tissue of the normal group rats was relative expression. The average daily energy intake, body weight, body length, Lee's index, body fat ratio, blood pressure, fasting blood glucose, triglyceride, free fatty acid, leptin, lipoprotein, Ghrelin, Obestatin, and leptin gene expression in adipose tissue were expressed at all times. There was no statistical difference.2. The effect of different dietary fat supply ratio and productivity structure on intestinal microflora structure of rats under free feeding state tenth weeks compared with zeroth weeks, the intestinal microflora of rats showed the decrease of bacteriobacteria, the increase of the thick wall bacteria gate and the increase of deformable bacteria doors. Tenth weeks, the abundance and diversity of intestinal microflora among the groups There was no difference in sex. The relative abundance of bacilli was the lowest in the intestinal flora of the low fat low protein group, and the thicker wall and the low fat and high protein group, the low carbon water group, the high fat and low carbon water group were significantly higher, the ratio of the bacilli / thick wall bacteria was lower than the low fat high protein group and the medium fat low carbon water group, and the intestinal microflora of the low fat and high carbon water group was S24-7 The relative abundance was higher than that in the low fat and low protein group; the low fat and high protein group Defluviitaleaceae was higher than the high fat and low carbon water group; the VadinBB60 in the low fat and low protein group was higher than all the groups except the high fat and low protein group; the low fat and high carbon water group Incertae_Sedis was higher than the middle fat and low carbon water group and the high fat and low carbon water group. Conclusion: 1, the different dietary fat supply ratio and the productivity nutrients junction are in conclusion. The Lee's index, body fat ratio, blood pressure, blood pressure, blood sugar, triglyceride, free fatty acid, leptin, adiponectin, Ghrelin, Obestatin, and the expression of leptin gene in adipose tissue have no influence on the.2. The high fat supply can cause the decrease of high density lipoprotein cholesterol and the production of insulin resistance, which may make the rats lose weight. The high carbohydrate supply can improve the expression of the adiponectin gene in the state of.3, which can increase the metabolic disorder of glycolipid. The specific mechanism still needs to further study the.4. Under the free feeding state, the abundance and diversity of the intestinal flora of rats have no effect on the abundance and diversity of the intestinal flora, and the high fat diet is higher than the high fat diet, compared with the high fat diet. Carbohydrate supply is more conducive to the proliferation of S24-7, D) efluviitaleaceae and incertae_Sedis than dietary, and the specific biological significance remains to be further studied.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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