【作者】 蔡滢；

【导师】 田德润；

【作者基本信息】 天津医科大学 ， 人体解剖与组织胚胎学， 2011， 博士

【摘要】 随着人们生活水平的提高,肥胖人口在全球范围内不断增多,肥胖诱发的相关疾病也日趋严重,威胁着人类的健康,加重了社会负担。肥胖的产生主要是由于摄入的热量过多,在体内形成脂肪堆积造成的。在维持机体能量平衡的过程中,下丘脑发挥了关键的作用。前期研究发现,下丘脑能够释放具有刺激或抑制摄食作用的神经多肽,如神经肽Y (NPY)和a-黑色素细胞刺激促激素(a-MSH)等,通过调控摄食行为维持机体能量平衡。随着研究的深入,不断有新的信号蛋白被发现对肥胖的产生发挥了重要作用,以往的理论已不能完全解释肥胖产生的原因。目的：本研究旨在通过对DIO和DR大鼠下丘脑的蛋白组学研究,筛选并鉴定下丘脑差异表达的蛋白质,揭示下丘脑内摄食相关蛋白质和神经肽在肥胖发展过程的作用,为肥胖的防治提供新的靶标。方法：1.建立DIO-DR大鼠模型。2.采用双向凝胶电泳技术(two-dimensional gel electrophoresis,2DE)结合基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS)的技术筛选并鉴定DIO-DR大鼠下丘脑差异表达的蛋白质。3.进一步运用荧光差异双向电泳(two-dimensional differential in-gel eletrophoresis,2D-DIGE)结合串联质谱(MALDI-TOF-MS/MS)的技术筛选并鉴定DIO-DR大鼠下丘脑差异表达的蛋白质。4.利用免疫组化和western blotting的方法对DIO-DR大鼠下丘脑部分差异表达蛋白质(包括PDI、GSTM1、SNAP-25等)进行定位和定量的检测,并对其功能展开深入研究。结果：.DIO和DR大鼠表型差异：1.DIO大鼠与DR大鼠初始体重相当,给予高脂饮食后,DIO大鼠体重增长速度快于DR大鼠,每日平均摄食量多于DR大鼠。2.DIO大鼠腓肠肌、睾丸和心脏的重量与DR大鼠相当,而内脏脂肪含量如附睾、肾周脂肪重量明显高于DR大鼠。3.DIO大鼠的甘油三酯、自由脂肪酸水平高于DR大鼠,而DR大鼠与普通饲料对照组水平相当。4. Elisa检测发现在肥胖早期(4周)和中期(8周)DIO大鼠的血清瘦素、胰岛素水平与对照组和DR组相比没有明显差异；而在肥胖后期(14周)DIO大鼠血清瘦素、胰岛素水平明显高于DR组(p<0.05),DR大鼠水平与对照组无显著性差异,提示DIO大鼠长期高脂喂养后出现瘦素抵抗和胰岛素抵抗。二.2DE结合质谱技术对DIO-DR大鼠下丘脑进行差异表达蛋白质的筛选和鉴定：1.筛选并鉴定出22种蛋白质,其中7种在DIO组呈高表达,7种下调,6种在DR组高表达,6种下调。2.这22种蛋白质分别属于抗氧化蛋白(PRDX1, GSTM1)、细胞代谢相关蛋白(UCHL-1X)、细胞周期调控蛋白、细胞骨架蛋白及未知蛋白等不同类别。三.2D-DIGE结合串联质谱对DIO-DR大鼠下丘脑进行差异表达蛋白质的筛选和鉴定：1.运用2D-DIGE筛选出42个蛋白质点,运用串联质谱技术(MALDI-TOF-MS/MS)鉴定出33种蛋白质,其中DIO组与对照组相比表达上调的有9个,下调的有2个；DIO组与DR组相比表达上调的有8个,下调的有9个；DR组与对照组相比上调的有8个,下调的有4个。2.将2D-DIGE结合串联质谱技术与2DE结合质谱技术进行比较发现,2D-DIGE所需样品量少,敏感性和特异性更高,筛选出更多的差异蛋白质点。而串联质谱技术也比一级质谱鉴定出更准确的蛋白质结构,得到更信服的结果。这33个蛋白质其中部分与2DE结合一级质谱的鉴定结果相一致,主要包括抗氧化蛋白、分子伴侣、细胞代谢相关蛋白、突触相关蛋白、细胞骨架蛋白等几个类别,其中抗氧化蛋白1(PRDX1X)、谷胱甘肽转移酶1(GSTM1X)、二硫键异构酶(PDI)、泛素C末端水解酶L1 (UCHL1)等可能与肥胖的发生有密切的联系,并发现了动力蛋白1(Dynamin 1)和SNAP-25等新的信号蛋白。四.免疫组化和western boltting方法对差异蛋白质进行定位和定量的研究1.免疫组化发现PDI、PRDX1和Dynamin1在DIO大鼠下丘脑阳性表达增强；SNAP-25在DIO大鼠下丘脑表达下调,在DR大鼠下丘脑表达上调。2. western blotting检测发现Dynamin1在DIO组表达上调,与对照组和DR组相比具有显著性差异；PDI、PRDXl和GSTM1在DIO组表达上调且在DR组表达下调(与对照组相比);UCHL-1和SNAP-25在DIO组表达下调且在DR组表达上调(与对照组相比)。结论：1.成功建立了DIO-DR大鼠模型,与人类肥胖具有很多相似的特征。2.通过蛋白质组学的研究,成功筛选和鉴定了DIO和DR大鼠下丘脑差异表达蛋白,而DIGE技术的应用,发掘了更多肥胖相关的信号分子,为深入研究肥胖的发病机制奠定了基础。3.DIO大鼠下丘脑由于长期高脂饮食发生细胞应激反应(氧化应激和内质网应激),干扰了瘦素和胰岛素的信号转导通路,造成DIO大鼠瘦素抵抗和胰岛素抵抗,并进而发展为肥胖。更多还原

【Abstract】 The number of obese people has increased rapidly all over the world for the reason of an increase in food availability. Obese subjects have an increased risk of developing comorbidities and lie heavy upon society. Obesity is a complex disease mainly caused by an increase in body fat mass resulting from an imbalance between energy intake and expenditure. Hypothalamus plays an crucial role in regulating the energy homeostasis of the body. Previous research has found that hypothalamus could release orexia peptides and anorexia peptides such as neuropeptide Y and a-MSH to maintain the balance of energy by regulating food intake. By the development of proteomic approaches, more and more new proteins related to obesity have been found which play key roles in obesity.Objective:Our study aims at selecting and identifying differential expressed proteins of hypothalamus in DIO-DR rats by proteomic analysis, in order to illuminate the effect of proteins and neuropeptides during the progress of obesity and provide new target of therapy in the future.Methods:1. Build DIO-DR model.2. The two-dimensional gel electrophoresis was performed to separate differential expressed proteins of hypothalamus in DIO-DR rat. The differential proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry.3. Use two-dimensional differential in-gel eletrophoresis and MALDI-TOF-MS/MS to separate and identify differential expressed proteins of hypothalamus in DIO-DR rat.4. The partial proteins including PDI, GSTM1 and SNAP-25 were validated by immunohistochemistry and western blotting.Results:1. Phenotype differentiations between DIO and DR rats.1) Although initial body weights were similar, DIO rats grew faster than DR rats did during the high fat fed period. DIO rats also took in more energy per day than DR rats did.2) Weight of main organs, including heart, gastrocnemius muscle and testes were similar between DIO and DR rats, but weight of visceral fats in DIO rats including fats of epididymis and around kidneys were significantly higher than that in DR rats.3) Plasma triglycerides and free fatty acid (FFA) level were higher in DIO rats than in DR rats.4) Serum level of insulin and leptin were measured by Elisa in different period. We found no significant difference between DIO and DR rats in early stage (4 weeks) and middle stage (8 weeks). But in later stage (14 weeks), insulin and leptin were significantly increased in DIO rats, and no significant difference between DR and control rats. This result suggests that DIO rats have insulin-resistant and leptin-resistant in the later stage.2. Separate and identify differential proteins of hypothalamus by 2DE integrated with MALDI-TOF-MS.1) 22 differential proteins were identified,7 of them were over expressed and 7 were down expressed in DIO rats; 6 of them were over expressed and 6 were down expressed in DR rats.2) These identified proteins were classified into antioxidant proteins (PRDX1, GSTM1), metabolism enzymes (UCHL-1), modulins of cell cycle, cytoskeleton proteins and unknown proteins.3. Separate and identify differential proteins of hypothalamus by 2D-DIGE integrated with MALDI-TOF-MS/MS.1) 42 differential protein spots were separated by 2D-DIGE and then 33 proteins were identified by MALDI-TOF-MS/MS.9 of them were up-regulated and 2 were down-regulated in DIO rats compared to control; 8 were up-regulated and 9 were down-regulated in DIO compared to DR; 8 were up-regulated and 4 were down-regulated in DR compared to control.2) 2D-DIGE is more sensible and specific than 2DE and can separate more protein spots. MALDI-TOF-MS/MS has more accuracy than MALDI-TOF-MS. Some of 33 identified proteins were same as which identified by 2DE and MALDI-TOF-MS. These proteins were classified into antioxidant proteins, molecular chaperon, metabolism enzymes, synapse associated proteins and cytoskeleton proteins. Some of them such as PRDⅪ, GSTM1, PDI and UCHL-1 have close relationship with development of obesity. We also found some new proteins such as Dynamin 1 and SNAP-25 which may be related with obesity.4. Partial differential proteins were validated by immunohistochemistry and western blotting.1) The data of immunohistochemistry assay showed PDI, PRDⅪand Dynaminl were over expressed as well as SNAP-25 was down regulated in hypothalamus of DIO rats.2) The results of western blotting demonstrated PDI, PRDⅪ, GSTM1 and Dynaminl were significantly over expressed in DIO rats (p<0.05) as well as PDI, PRDⅪ, GSTM1 were down regulated in DR rats. On the contrary, UCHL-1 and SNAP-25 were over expressed in DR rats and down regulated in DIO rats.Conclusions:1. DIO-DR rat model was built up successfully which has many characteristics similar to that of human being.2. Differential proteins were separated and identified successfully by 2D-DIGE integrated with MALDI-TOF-MS/MS. More signaling proteins relating to obesity have been selected by 2D-DIGE, which makes foundation for further research.3. Cellular stress (oxidative stress and ER stress) in hypothalamus of DIO rats induces insulin-resistant and leptin-resistant by interfering signal transduction pathway of insulin and leptin due to chronic high fat diet.更多还原