【作者】 叶文慧；

【导师】 蔡德鸿；

【作者基本信息】 南方医科大学 ， 内分泌与代谢病， 2011， 硕士

【摘要】 研究背景近年来,由于物质生活水平的提高、体力劳动的减少和口老龄化进程加快,糖尿病在世界范围内的患病率呈急剧上升趋势,已经成为继心脑血管疾病、恶性肿瘤之后导致人类整体健康水平下降的罪魁祸首之一。目前全球拥有超过2.4亿人已经被诊断为糖尿病,专家预测到2025年,全球估计将有3.5亿人以上罹患糖尿病。2009年公布的“中国糖尿病及代谢综合征流行病学调查”的最新数据显示,20岁以上的城市、乡镇和富裕农村人群中,糖尿病患病率已达9.7%,另外还有15%的人处于糖调节异常阶段。如果不采取有效的防治手段,糖尿病可能成为21世纪人类将面临的一场新的灾难。果糖(Fructose, FRU)一般制成晶体或者糖浆作为商业用途。在过去的20年里,果糖作为甜味剂在加工食品及软性饮料中从20%增加至30%,这和同期剧烈上升的肥胖发生率相似。有研究提示,日常高脂饮食、喝大量果糖的软饮料所导致的超重和肥胖,与高果糖高脂导致的高甘油三酯血.症、代谢综合征、糖耐量受损、胰岛素抵抗的发生密切相关,但果糖如何导致甘油三酯增高、非酒精性脂肪肝,如何导致胰岛素抵抗、使机体出现胰岛素敏感性降低,继而出现糖耐量受损,其确切的分子机制尚未完全阐明。所以,对此作进一步的研究,阐明其机制并针对其发生机制采取相应的干预措施,对预防肥胖、糖尿病及代谢综合症的发病意义重大。而对其相关发病机制的进一步研究,就需要建立一种与现代人饮食模式相似、糖脂代谢亦与人类相似的动物模型。既往有文献指出：兔、大鼠或小鼠模型在脂质代谢方面均与人类有较大差别,小鼠血量少,不利于血生化分析及实验设计,用上述动物作为研究现代社会人类高糖高脂饮食的动物模型,具有一定的局限性。另一方面,有研究表明,叙利亚金黄地鼠,特别是雄性金黄地鼠,在糖脂代谢方面与人类最接近,可能是糖脂代谢紊乱建模的新选择。大量证据表明脂肪组织不仅是能量的储存所更是重要的内分泌器官,脂肪组织生成和释放许多生物活性分子,通过局部和全身作用,协调能量代谢、胰岛素敏感性、炎症和血管反应等。脂肪型脂肪酸结合蛋白(Adipocyte fatty acid binding protein, A-FABP)主要表达于脂肪细胞和巨噬细胞,它作为脂肪酸的转运蛋白,影响着脂肪酸的代谢及脂肪酸信号,与糖尿病和动脉粥样硬化密切相关。是成熟脂肪细胞中的量最大的蛋白,属于脂肪酸绑定蛋白家族中的一员,在组织细胞间脂肪酸运输及能量代谢中有重要作用。最近有动物模型研究提不A-FABP可能在糖自动调节机能中有重要作用,且发现糖代谢紊乱与A-FABP水平显著相关。本实验通过检测A-FABP在不同膳食组中的变化,探讨其与血糖升高、胰岛素抵抗、胰岛素敏感性降低之间的相关性。综上所述,本课题选择叙利亚金黄地鼠为研究对象,通过不同高脂膳食成分饲料诱导方式,建立更接近现代人膳食成分糖脂紊乱的动物模型,为目前糖尿病、肥胖、代谢综合征、高甘油三酯血症、非酒精性脂肪肝的发生机制及其早期干预研究提供动物模型。并同时了解在不同膳食喂养条件下A-FABP浓度变化,研究其与胰岛素抵抗的关系。研究目的以不同成分膳食喂养叙利亚金黄地鼠,通过设正常对照组、高脂组、高脂+葡萄糖组、高脂+果糖组,观测叙利亚金黄地鼠体重、血清糖脂水平、胰岛素、A-FABP浓度、肝指数及肝脏和胰腺组织形态变化。对比分析正常与病理状态下叙利亚金黄地鼠糖脂变化可能发生机制。旨在建立更接近现代人饮食习惯的糖脂代谢异常动物模型。研究方法1.随机选取12周健康雄性叙利亚金黄地鼠,分为四组,每组6只。正常对照组(N组)用普通饲料喂养,单纯高脂组(HF组)用单纯高脂饲料喂养,葡萄糖高脂组(HF+GLU组)用20%葡萄糖糖溶液+高脂饲料喂养,高脂果糖组(HF+FRU)用20%果糖溶液+高脂饲料喂养。共喂养12周,分别在第0、1、2、6、12周测体重、酶法测定金黄地鼠血清甘油三酯、胆固醇、血糖水平。2.12周末处死金黄地鼠,暴露腹腔,心脏取血3-4mL,室温下静置30min 3000rP离心15min,取血浆10μL分装保存于-80℃冰箱备检。肉眼观察肝及胰腺,肝脏称重,计算肝指数(肝指数=肝脏质量/体质量×100%)。留取肝脏叶、分离胰腺,立即投入10%甲醛溶液固定,逐级乙醇脱水,二甲苯透明,石蜡包埋切片,HE染色,光学显微镜下观察各组金黄地鼠肝组织学变化。3. ELISA法测定各组金黄地鼠血清胰岛素及A-FABP浓度：采用美国R&D公司大鼠血清A-FABP试剂盒及血清胰岛素试剂盒,即酶联免疫吸附法测定血清A-FABP及胰岛素浓度。严格按说明书操作,批内差异11%,批间差异10%。计算胰岛素敏感指数：胰岛素敏感指数ISI=LIN 1/(空腹血糖值FBG×空腹血清胰岛素值FIN)。4.统计学分析：采用SPSS 13.0统计软件进行分析,数据以X±s表示,多组比较方差齐采用one-way ANOVA,组间多重比较采用LSD法,不齐则采用Welch检验,多重比较采用Tamhane检验,不同时间指标比较采用重复测量方差分析。P<0.05认为差异有统计学意义。研究结果1.不同膳食成分各组叙利亚金黄地鼠的体重变化：各组体重水平均持续上升,12周时达最高峰。HF组、HF+GLU组、HF+FRU组与N组相比升幅更大(P<0.01)。各个时间点HF+FRU组体重均较高于其余三组(P<0.01)。2.不同膳食成分各组叙利亚金黄地鼠的血生化特点：甘油三酯：N组12周甘油三酯水平基本持平。HF组第2周显著升高,第6周时下降,到第12周显著上升。HF+GLU组呈持续上升趋势,12周达峰值。HF+FRU组在2周已达到一平台,12周时达最高峰。入组时四组甘油三酯水平无显著差异,不同膳食成分喂养后一周始,各个时间点均以HF+FRU组甘油三酯高于其余三组(P<0.01)。胆固醇：HF组、HF+GLU组及HF+FRU胆固醇均在1周后显著升高,HF+GLU组及HF组在2周达最高峰,6周及12周时稍有下降。HF+FRU组呈持续上升趋势,12周达峰值。HF组、HF+GLU组、HF+FRU组的胆固醇从入组时2mmol/L左右上升至12周的7.5mmol/L左右,而正常组则从基本维持在1.95左右。HF组、HF+GLU组、HF+FRU组胆固醇上升显著高于正常组(P<0.01)。血糖：正常组在研究的12周内血糖变化无统计学意义(P>0.05)。HF、HF+FRU、HF+GLU糖组与正常组血糖变化幅度不同,血糖最大值在12周,最小值在0周,在1周即明显升高,6周升高后出现平台,其中HF+FRU组血糖升高幅度比HF、HF+GLU组更显著(P<0.01)。3.各组金黄地鼠肝脏及胰腺组织形态学改变：肝脏：肉眼：正常组肝脏无异常变化;Hf组及HF+GLU组肝脏颜色黄紫红色相间,边缘钝,部分肝脏表面呈沙粒样改变,体积增大；HF+FRU组地鼠肝脏呈黄白褐色,肝脏体积显著增大,边缘圆钝,包膜紧张。光镜：正常对照组形态未见明显变化。HF组及HF+GLU组,肝细胞明显肿胀增大,有大量脂肪滴存在,部分肝细胞呈空泡状,肝中央静脉和小叶间静脉含有少量红细胞,肝细胞间见炎性细胞；HF+FRU组与HF及HF+GLU组相比较脂肪样变更显著,脂肪滴弥漫累积大多数小叶,细胞核深染,细胞明显肿胀,肝细胞排列不整齐,见大量炎性细胞,并可见脂肪囊形成。胰腺：肉眼未见明显差异,光镜：正常对照组,胰岛大小数量正常,大小均一,形状规则,界限清楚；HF组、HF+GLU组,HF+FRU组胰岛增生,体积普遍增大,数量增多,但三组间差别不显著。肝指数：与正常组相比,HF组、HF+GLU组、HF+FRU组肝指数(肝指数=肝脏质量/体质量×100%)明显升高,差异有显著性(P<0.01)；与HF组、HF+GLU组比较,HF+GLU组肝指数水平升高更显著(P<0.01)。4.胰岛素及胰岛素敏感指数：血清胰岛素：HF组、HF+GLU组、HF+FRU组的血清胰岛素水平均高于正常对照组,与HF及HF+GLU组相比,HF+FRU组胰岛素水平升高更显著(P<0.01)。胰岛素敏感指数：胰岛素敏感指数[ISI=LIN 1/(空腹血糖值FBG×空腹血清胰岛素值FIN)]:HF组、HF+GLU组、HF+FRU组均低于N组,差异具有显著性(P<0.01),其中HF+FRU组与HF组、HF+GLU组相比,胰岛素敏感指数更低,差异具有显著性(P<0.01)。6.血清A-FABP:血清A-FABP:四组金黄地鼠血清A-FABP水平,HF组、HF+GLU组、HF+FRU组血清A-FABP浓度均高于N组,差异具有显著性。其中HF+FRU组与HF组、HF+GLU组相比,血清A-FABP浓度更高,差异具有显著性,P值均<0.01。研究结论1.经一定周期不同成分的高脂饲料喂养,叙利亚金黄地鼠可形成典型的血脂紊乱、高糖血症、胰岛素抵抗、非酒精性脂肪肝及胰岛增生。其中,高脂高果糖金黄地鼠模型组与高脂模型组及高脂高葡萄糖模型组相比较,甘油三酯、胆固醇、血糖升高、胰岛素分泌增多、胰岛素敏感性降低更明显,非酒精性脂肪肝及胰岛增生现象更显著。2.高脂高果糖雄性金黄地鼠模型的膳食成分更好地模拟人类饮食结构及人类糖脂代谢紊乱的病理过程,为现代人高脂高果糖饮食所导致糖耐量受损、高脂血脂、代谢综合征的研究提供更有优势的实验动物模型。3.血清A-FABP水平在胰岛素分泌增高、胰岛素敏感性降低更显著的高脂高果糖地鼠组中显著增加,血清A-FABP可能是糖尿病、代谢综合征的发生、发展一个标志物。更多还原

【Abstract】 BackgroundIn recent years, as the improvement of living standards, reducing labor and increasing aging population the proportion of diabetes in the world showed a sharp rise in the prevalence of the trend of diabetes. Diabetes has become one of the culprit of malignant tumors which decline hunman health level, that after heart and cerebrovascular diseases. Currently more than 240 million people around the world have been diagnosed with diabetes, experts predict that in 2025, there is estimated to be more than 3.5 million people suffer from diabetes. What published in 2009 saying, "China epidemiological survey of diabetes and metabolic syndrome, the latest data show that over the age of 20 cities, towns and prosperous rural population, diabetes prevalence has reached 9.7%, plus 15% in glucose regulation stage. If do not take effective means for prevention and treatment, diabetes may become the 21st century new disaster.Fructose (Fructose, FRU) generally made of crystal or syrup for commercial purposes. In the past 20 years, as a sweetener in processed foods and soft drinks fructose increased from 20% to 30%, which increase similar to is the rate of increasesing obesity over the same period. Some studies suggest that daily high-fat diet, drink plenty of soft drinks, fructose caused overweight and obesity, high fat and high fructose induced hypertriglyceridemia is closely related to metabolic syndrome, impaired glucose tolerance, insulin resistance. However, how higher fructose cause triglyceride, non-alcoholic fatty liver disease, and insulin resistance, insulin sensitivity which was followed by impaired glucose tolerance, the related molecular mechanism has not been fully elucidated. Therefore, this study further clarify the mechanism and the mechanism for its take appropriate interventions to prevent obesity, diabetes and metabolic syndrome is of great significance. And their further study of the pathogenesis related to a need for a modern dietary patterns with similar glucose and lipid metabolism are similar to animal and human models. With previous literature suggests, rabbit, rat or mouse models of human lipid metabolism are very different, less blood in mice, is not conducive to biochemical analysis and experimental design, the use of such animals as the study of modern society, human high- sugar high-fat diet in animal models, with some limitations. On the other hand, studies have shown that Syrian hamster, especially the male hamster, glucose and lipid metabolism in the nearest human, therefore, this study is more similar to human lipid metabolism in the golden hamster as experimental animals, give 12 weeks of the different components of the high fat, sugar fat diet, observed hamster body weight, serum glucose and lipid metabolism, insulin, serum lipid fatty acid binding protein, the insulin sensitivity, liver index, liver and observed pathological changes of pancreatic tissue. Look closer to human due to the high sugar and fat diet caused abnormal lipid metabolism and insulin resistance in animal models. As obesity, metabolic syndrome, hypertriglyceridemia, diabetes, non-alcoholic fatty liver mechanism and early intervention (such as drug screening, nutritional factors, dietary intervention, etc.) to provide a new animal model.Considerable evidence that adipose tissue is not only the energy storage is more important endocrine organ, releasing a large number of biologically active substances into the blood stream. These adipose tissue-derived bioactive molecules, through the local and systemic role in coordinating energy metabolism, insulin sensitivity, inflammation and vascular response. Fat-type fatty acid binding protein (Adipocyte fatty acid binding protein, A-FABP) expressed mainly in fat cells and macrophages, as fatty acid transport protein, affecting the metabolism of fatty acids and fatty acid signal, with diabetes and atherosclerosis are closely related. Mature fat cells is the largest protein [2], fatty acid binding protein is a member of the family, between the fatty acid transport in cells and plays an important role in energy metabolism [3,4]. Recently, animal models suggest A-FABP may function in the automatic adjustment of sugar have an important role. In recent years, studies have found that glucose metabolism and A-FABP levels were significantly related. In this study, by detecting the A-FABP in different dietary groups, and to explore with the blood sugar, insulin resistance, insulin sensitivity, the correlation between reduced.In summary, our study group selected Syrian hamster as the research object, feed ingredients by high fat diet induced in different ways, the establishment of the modern dietary components closer to the animal model of lipid disorders, the current diabetes, obesity, metabolic syndrome, hypertriglyceridemia, non-alcoholic fatty liver disease pathogenesis and animal models of early intervention research. And also understand the feeding conditions in the different dietary concentrations of A-FABP was studied with insulin resistance.Purpose1. Through different components of diet to fed Syrian golden hamsters, establish a animal model which is close to modern human diet, the Syrian hamster is fat and high fructose, fatty liver, lipid metabolism model, and compare different diet components fed to the golden Hamster model of glucose and lipid metabolism. 2. To explore the normal control group (N), high-fat group (HF), high fat+ glucose group (HF+GLU), high fat+fructose group (HF+FRU).Check the Syrian hamster body weight, serum glucose and lipid levels, Insulin, A-FABP levels, liver index and morphological changes in liver and pancreas. Compara and analysis the normal and pathological conditions changes the Syrian hamster and its glycolipids possible mechanism.Methods1. Randomly selected 12 male Syrian golden hamsters, divided into four groups, n= 6. The normal control group (fed with normal diet), pure fat group (fed with high fat diet alone), glucose fat group (with 20% glucose solution+high fat diet sugar), fructose, high-fat group (with 20% fructose solution+high fat diet). Were fed for 12 weeks, respectively, measured at 0,1,2,6,12 weeks, body weight, enzymatic determination of hamster serum triglycerides, cholesterol, blood sugar levels.2.12 over the weekend killed hamster, exposure to the abdominal cavity, heart blood 3-4mL, room temperature for 30min 3000rP Centrifuge 15min, the plasma stored at -80℃10μL refrigerator equipment seized. Visual observation of liver and pancreas, liver weight, liver index (HSI=liver mass/body mass×100%). Specimens from the liver lobe, separating the pancreas,10% formaldehyde solution immediately put into a fixed, sequential ethanol dehydration, xylene, paraffin embedded sections, HE staining were observed under light microscope hamster liver.3.ELISA method hamsters in each group serum concentrations of insulin and A-FABP:American R & D companies in serum A-FABP and serum insulin kit kit, in strict accordance with manual operation,11% of intra differences, differences between batches 10%. Using enzyme-linked immunosorbent assay of serum A-FABP and insulin concentrations. Insulin sensitivity index:insulin sensitivity index ISI= LIN 1/(fasting plasma glucose FBG×fasting insulin levels FIN). 4. Statistical analysis:SPSS 13.0 statistical software to analyze data toⅩ(-)±s that homogeneity of variance with multiple comparison one-way ANOVA, multiple comparisons between groups using LSD method, missing the test with Welch, by Tamhane multiple comparison test was used to compare different time indices repeated measures analysis of variance. P<0.05 considered significant difference.Results1. Fed different dietary components Syrian golden hamsters in each group changes of body weight:The average body weight of water continued to rise, reached the peak at 12 weeks. HF group, HF+GLU group, HF+FRU group compared with the increase of N greater (P<0.01). Each time point compared with HF+FRU body weight than the other three groups (P<0.01).2. Composition of different dietary groups fed Syrian golden hamsters of the blood biochemical characteristics:TG:N Group 12-week triglyceride levels flat. HF group was significantly higher 2 weeks,6 weeks down to 12 weeks increased significantly. HF+GLU group has continued to rise,12 Zhou Dafeng value. HF+FRU group increased at 2 weeks Da Houda to a platform, reached the peak at 12 weeks. Group into four groups no significant difference in triglyceride levels, one week after feeding different dietary ingredients before, each time point are HF+FRU triglyceride was higher than other three groups (P<0.01).Cholesterol:HF group, HF+GLU group and HF+FRU cholesterol were significantly increased after 1 week, HF+GLU group and HF group reached a peak in 2 weeks,6 weeks and 12 weeks fell slightly. HF+FRU group process continued upward trend,12 Zhou Dafeng value. HF group, HF+GLU group, HF+FRU group into the group of cholesterol from 2mmol/L up to 12 weeks is about 7.5mmol/L, while the normal group from remaining at 1.95 or so. HF group, HF+GLU group, HF+FRU group of cholesterol increased significantly in the normal group (P<0.01).Glucose:normal groups during the 12 weeks no significant changes in blood glucose (P> 0.05). HF, HF+FRU, HF+GLU group and normal glucose range of different levels of blood glucose changes, blood glucose in the 12 weeks maximum, minimum,0 weeks, which significantly increased at 1 week,6 weeks after elevated platform, where the HF+FRU group than in blood sugar range HF, HF+GLU group more significantly (P<0.01).3. Hamsters in each group morphological changes in liver and pancreas:Liver:the naked eye:normal no abnormal changes in liver; HF group and HF+ GLU group of liver color yellow purple intersection, blunt the edge of some sand-like surface of the liver changes, the volume increases; HF+FRU group of hamster liver was yellow and white brown, liver volume increased significantly, blunt edge, coated tension. Light microscopy:the normal control group, no significant morphological changes. HF group and HF+GLU group, swelling of liver cells was increased, there is a large number of lipid droplets, some vacuolization of liver cells, liver interlobular vein central vein and a small amount of red blood cells, liver cells, see inflammatory cells; HF+FRU group and the HF and HF+GLU group compared to significant changes in fat-like, fat droplets accumulate most of the diffuse lobular nuclei stained cells were significantly swollen, irregular arrangement of hepatic cells, see a large number of inflammatory cells, and capsule formation of visible fat.Pancreas:no significant difference in the naked eye, light microscopy:the normal control group, the number of normal islet size, size uniformity, shape rules, clear boundaries; HF group, HF+GLU group, HF+FRU group of islet hyperplasia, the volume generally increases, the number of increased, but differences among the three groups was not significant. 4. Liver Index:Compared with normal group, HF group, HF+GLU group, HF +FRU group, the liver index:(liver index=liver mass/body mass Water 100%) was significantly higher, the difference was significant (P<0.01); with the HF group, HF +GLU group, HF+GLU group more significantly elevated levels of liver index (P <0.01).5. Insulin and insulin sensitivity index:Serum insulin:HF group, HF+GLU group, HF+FRU in the serum insulin levels were higher than the control group, with HF and HF+GLU group, HF+FRU group more significantly elevated insulin levels (P<0.01.) Insulin sensitivity index:insulin sensitivity index [ISI=LIN 1/(fasting plasma glucose FBG×fasting serum insulin FIN)]:HF group, HF+GLU group, HF+FRU group were lower than the N group, the difference was significant (P<0.01), in which HF+FRU group and HF group, HF+GLU group, a lower insulin sensitivity index, the difference was significant (P<0.01).6. Serum A-FABP:Serum A-FABP:four hamster serum A-FABP levels, HF group, HF+GLU group, HF+FRU serum A-FABP concentrations were higher than N group, the difference was significant. One group and HF HF+FRU group, HF+GLU group, higher serum A-FABP levels, the difference was significant, P values were<0.01.Conclusion1. After a certain period of different components of the high fat diet, the Syrian hamster can form the typical dyslipidemia, hyperglycemia, insulin resistance, non-alcoholic fatty liver and islet hyperplasia. Among them, high-fat high-fructose hamster model group and model group and model group, high fat compared with high glucose, triglycerides, cholesterol, blood sugar, increase insulin secretion, insulin sensitivity significantly decreased, non-alcoholic fatty liver and more significant phenomenon of islet hyperplasia.2. High-fat and high fructose male hamster model can simulate the human dietary ingredients and dietary glucose and lipid metabolism disorders of human pathological process better, the modern high-fat high-fructose diet caused impaired glucose tolerance, fat lipids, research of metabolic syndrome advantage of the experimental animal models.3. Serum A-FABP levels increased in group which insulin secretion increase, insulin sensitivity decrease, reduce fat and high fructose more significant to a significant increase in the rat group, the serum A-FABP may be diabetes, metabolic syndrome, the occurrence and development of a marker.更多还原