色氨酸耗竭模型大鼠的比较蛋白质组学研究

发布时间：2018-06-24 10:37

本文选题：抑郁症 + 色氨酸耗竭　；参考：《重庆医科大学》2012年硕士论文

【摘要】：背景抑郁症是一种持久的心境低落状态，其核心症状为情绪低落、兴趣缺乏、乐趣丧失、并伴有焦虑、自责自罪、自杀行为及睡眠障碍等。抑郁症已经成为当今世界危害人类健康最大的疾病之一，，其终身患病率为5%~17%。抑郁症的致病机制目前仍然不明确，且缺乏客观的诊断指标。选择性5-HT再摄取抑制剂（SSRIs）类抗抑郁药在临床上取得的疗效提示，抑郁症的发病与5-HT系统功能密切相关。色氨酸耗竭试验通过耗竭体外摄入的色氨酸，影响外周及中枢系统中5-HT的含量及功能，是研究5-HT系统改变的有效方法。建立色氨酸耗竭模型，并对模型动物的脑组织进行蛋白质组学分析的研究还未见报道。目的本课题将基础研究与临床医疗相结合，遵循转化医学的研究思路，通过建立大鼠快速色氨酸耗竭模型，分析5-羟色胺能系统与动物抑郁样行为发生的联系，并运用比较蛋白质组学结合质谱技术对模型动物的海马组织进行分离鉴定，旨在从机体蛋白质层面探究疾病的发病机制，为临床抑郁症诊断做一有益探索。方法本课题共分为三部分：第一部分，大鼠快速色氨酸耗竭模型的建立与评价；第二部分，大鼠快速色氨酸耗竭模型组织的比较蛋白质组学研究。雄性Sprague-Dawley大鼠，体重300g~350g，行为学筛选后随机分为三组：快速色氨酸耗竭模型组（TRP-组），含色氨酸的胶原蛋白对照组（TRP+组），盐水对照组（Saline组）。适应性灌胃10天后进行常规灌胃造模，采用糖水消耗实验和敞箱实验对动物进行行为学检测，运用高效液相色谱技术检测分析造模后动物血浆中的氨基酸含量。选择与抑郁症发病相关的海马区进行单个样本的比较蛋白质组学分析。三氯乙酸-丙酮沉淀法提取组织蛋白，结合双向电泳分离技术，ImageMaster2D Platinum6.0软件对2-DE图像进行分析，MALDI-TOF-MS质谱鉴定差异蛋白，所得质谱数据在NCBInr数据库中检索蛋白相关信息，分析蛋白功能。结果造模后，各组动物的饮水量均有所下降，其中TRP-a组的糖水偏嗜度显著低于Saline组和TRP+组（P均0.01），与其自身的基线数据也有显著差异（P0.05）。TRP-a组动物活动的总路程显著低于Saline组和TRP+组（P均0.05），与其自身的基线数据也有显著差异（P0.05）。动物造模后氨基酸检测结果显示，与Saline组和TRP+组相比，快速色氨酸耗竭模型组（TRP-组）均表现出色氨酸含量明显下降，且具有显著性差异（P0.001）；与Saline组和TRP+组相比，观察指标TRP/∑LNAA在TRP-组也表现出显著性差异（P0.001）。 ImageMaster2D Platinum6.0软件对2-DE图像进行分析，各组间两两比较，共分析出88个差异表达的蛋白质（%Vol1，P0.05），并对其中14个差异蛋白进行了质谱鉴定。差异蛋白基本分为以下6类：（1）细胞骨架相关蛋白：如Septin-11基因编码的蛋白；（2）神经发育相关蛋白：如二氢嘧啶酶相关3、髓鞘碱性蛋白；（3）信号转导相关蛋白：如生长因子受体结合蛋白2、Stathmin；（4）能量代谢相关蛋白：如NADH脱氢酶、H+-ATPase；（5）转录和调控相关因子：如Sirtuin2、泛素结合酶E2N、受翻译调节的肿瘤蛋白；（6）氧化代谢相关蛋白：如氨基酰化酶-1A、谷胱甘肽转移酶Mu1、3-磷酸甘油脱氢酶。结论高效液相色谱检测血浆氨基酸数据表明，动物模型成功耗竭了外周血中的色氨酸，且由TRP/∑LNAA指标可知，由外周血浆进入中枢神经系统的色氨酸量也明显降低，并推测由色氨酸生成的5-HT量也会明显降低。通过动物行为学评价，快速色氨酸耗竭试验能模拟人类5-TH能系统改变所导致的抑郁情绪、快感缺失、活动减少等情绪。模型操作简单、安全、可逆，具备可行性。检索文献，分析差异蛋白功能，发现其对抑郁症的相关研究有重要意义。
[Abstract]:background
Depression is a persistent state of depression, its core symptoms are low mood, lack of interest, loss of fun, and anxiety, self blame, suicide and sleep disorders. Depression has become one of the most harmful diseases in the world today, and its life-long prevalence rate is still the pathogenesis of 5%~17%. depression. It is not clear and lacks objective diagnostic indicators. The clinical efficacy of selective 5-HT reuptake inhibitor (SSRIs) antidepressants suggests that the pathogenesis of depression is closely related to the function of the 5-HT system. The depletion test of tryptophan exhaustion tests the content and function of 5-HT in the peripheral and central systems by consuming tryptophan in vitro and in the central nervous system. To investigate the effective way to change the 5-HT system, a tryptophan depletion model was established, and the proteomic analysis of the brain tissues of the model animals was not reported.
objective
This topic combines basic research with clinical medical treatment and follows the research ideas of transformation medicine. By establishing rat fast tryptophan depletion model, the relationship between 5- serotonin energy system and animal depressive behavior is analyzed, and the hippocampus tissue of model animals is identified by comparative proteomics combined mass spectrometry. Explore the pathogenesis of the disease from the protein level of the body, so as to make a useful exploration for the diagnosis of clinical depression.
Method
This topic is divided into three parts: the first part, the establishment and evaluation of rat fast tryptophan exhaustion model; the second part, the comparative proteomics study of the rat model of rapid tryptophan depletion model.
Male Sprague-Dawley rats and body weight 300g~350g were randomly divided into three groups: fast tryptophan exhaustion model group (group TRP-), tryptophan collagen control group (group TRP+) and saline control group (group Saline). Routine gavage was performed for 10 days after adaptive gavage, and the animals were carried out using sugar water consumption experiment and open box experiment. The content of amino acids in animal plasma was detected by high performance liquid chromatography (HPLC).
A comparative proteomic analysis of a single sample of the hippocampus associated with depression was selected. Three chloroacetic acid acetone precipitation method was used to extract tissue protein, combined with two-dimensional electrophoresis separation technology, ImageMaster2D Platinum6.0 software was used to analyze 2-DE images, MALDI-TOF-MS mass spectrometry was used to determine the difference protein, and the mass spectrum data were in the NCBInr database The protein related information was retrieved and the function of protein was analyzed.
Result
After the model, the amount of drinking water in each group decreased, and the sugar and water bias of group TRP-a was significantly lower than that of group Saline and TRP+ (P 0.01), and there was significant difference with its own baseline data (P0.05) the total route of animal activity in group.TRP-a was significantly lower than that of group Saline and TRP+ group (P 0.05), and there were significant differences with their own baseline data (P0). .05). After the animal model, the results of amino acid detection showed that the rapid tryptophan depletion model group (TRP- group) showed significant decrease in ammonia acid content and significant difference compared with group Saline and TRP+ group (P0.001). Compared with group Saline and TRP+ group, the observation index TRP/ Sigma LNAA showed significant difference in TRP- group (P0.001).
ImageMaster2D Platinum6.0 software analyzed the 2-DE images. 22 groups were compared. 88 differentially expressed proteins (%Vol1, P0.05) were analyzed, and 14 of them were identified by mass spectrometry. The difference proteins were divided into 6 categories: (1) cytoskeleton related proteins, such as the protein encoded by Septin-11; (2) nerve Development related proteins: two pyrimidase related 3, myelin basic protein; (3) signal transduction related proteins, such as growth factor receptor binding protein 2, Stathmin; (4) energy metabolism related proteins, such as NADH dehydrogenase, H+-ATPase; (5) transcription and regulation related factors: Sirtuin2, ubiquitin binding enzyme E2N, and translators regulated tumor proteins (6) oxidative metabolism related proteins such as aminoacylase -1A, glutathione transferase Mu1,3- glycerol phosphate dehydrogenase.
conclusion
The determination of plasma amino acid data by high performance liquid chromatography shows that the animal model has successfully exhausted the tryptophan in the peripheral blood, and from the TRP/ Sigma LNAA index, the tryptophan amount from the peripheral plasma into the central nervous system is also significantly reduced, and the amount of 5-HT generated by tryptophan also decreases obviously. The ammonia depletion test can simulate the depressive mood, the loss of pleasure, and the decrease of activity caused by the human 5-TH system changes. The model is simple, safe, reversible, and feasible.
Retrieving the literature and analyzing the function of differentially expressed proteins, it is found that it is of great significance to the study of depression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.4

【参考文献】