基于iTRAQ技术的电针抗抑郁有效靶点筛选研究

发布时间：2018-04-27 05:26

本文选题：差异蛋白 + 电针　；参考：《北京中医药大学》2015年博士论文

【摘要】：背景与目的近年来,抑郁症与药物成瘾、中枢神经系统疾病成为全球公共健康关注的主要问题。抑郁症与成瘾、中枢神经系统疾病的高共病性也逐渐成为关注的热点。目前抑郁症的病生理机制尚不清楚。但是电针治疗抑郁症的临床疗效已经得到了广泛的认可,具有疗效确切,整体调节结合多靶点治疗的特点,同时对药物的毒副作用也有减毒增效作用。电针抗抑郁机理研究已经备受关注,大量研究证实电针能从多角度、多靶点、多途径起到抗抑郁作用。人类绝大多数病理生理过程都是由蛋白质参与完成的,在疾病的发生、机体的反应以及疾病转归的每一个过程,蛋白质都发挥着极其重要的作用,因此,蛋白质作为生命活动的重要组成部分,蛋白标志物的筛选就成为科学家们关注的热点。因此从蛋白质水平探讨电针抗抑郁机制是十分必要的。关于致炎性细胞因子与抑郁症发病的关系已有较深入的研究,而转化生长因子(TGF-p)及突触结合蛋白(Syntaxin-1)与抑郁症的关系研究尚不多见。本实验基于同位素相对与绝对定量(iTRAQ)技术,联合多维液相色谱技术与串联质谱技术(LC-MS/MS)分离和鉴定差异蛋白,比较电针组与模型组海马组织蛋白定量结果,对电针所干预的慢性束缚应激大鼠的海马组织蛋白进行初筛,寻找电针抗抑郁的有效靶点,最后,选用Western blot技术对部分差异蛋白进行验证。方法本研究应用慢性束缚应激结合孤养建立抑郁大鼠模型,采用旷场实验、体质量变化情况来评价抑郁模型是否复制成功,并应用免疫组化技术对大鼠海马组织CA3区炎性细胞因子IL-1β、IL-6进行检测,确认大鼠是否出现免疫炎症反应；同时,采用免疫组化技术对大鼠海马组织CA3区不同于其他炎性因子的转化生长因子TGF-β、突触膜结合蛋白syntaxin-1阳性神经元的积分光密度(IOD)值进行检测；采用iTRAQ试剂盒标记海马组织蛋白肽段,经强阳离子交换柱(SCX)分离,之后使用Triple TOF 5600质谱仪对蛋白质多肽质谱图进行定量鉴定,经过Mascot软件分析后,描绘差异蛋白质谱。挑选出差异蛋白后,登陆Gene Ontology与大鼠数据库IPI-rat_3.87数据库进行差异蛋白质的基因和基因产物的属性分析；通过Cluster of Orthologous Groups of proteins (COG)数据库预测这些蛋白质可能的功能并对其功能做分类统计；通过Pathway分析与KEGG数据库确定差异蛋白参与的最主要的生化代谢途径和信号转导途径。经过生物信息学分析及文献查阅后,挑选出部分差异蛋白,应用Western blot技术验证其表达水平。结果1.造模完成后,模型组大鼠旷场实验的水平运动次数、垂直竖立次数、体质量均显著低于对照组(P0.01)说明造模成功；与模型组相比,电针组旷场实验水平及体质量明显升高(P0.01)。2.采用免疫组化技术检测海马组织CA3区IL-1β和IL-6阳性神经元的IOD值。结果发现：模型组IL-1β、IL-6的表达与对照组相比明显增多(p0.01)；电针组与模型组比较有显著差异(p0.05),电针组阳性神经元数量少于模型组。从免疫组化图片来看,对照组大鼠海马CA3区阳性细胞数量较少且排列有序,胞浆着色较浅；模型组大鼠海马CA3区阳性细胞数量明显增多,排列整齐程度尚可,着色深；电针组大鼠海马CA3区阳性细胞数量较模型组少,且排列较有序,胞浆着色较模型组略浅。3.采用免疫组化技术检测海马组织CA3区TGF-β阳性神经元的IOD值。结果发现：从免疫组化图片来看对照组和电针组阳性细胞数量较少且着色较浅,排列较整齐。而模型组则分布有较多密集、着色较深的阳性细胞,且排列略不整齐。然而,慢性束缚应激结合孤养能够引起大鼠海马组织CA3区TGF-β表达较对照组明显升高(p0.01),但是电针干预后其表达与模型组相比却没有统计学意义(p0.05)。4.采用免疫组化技术检测海马组织CA3区syntaxin-1阳性神经元的IOD值。结果发现：经过28后,模型组海马CA3区syntaxin-1阳性神经元的表达与对照组和电针组比较明显下降(p0.01)。从免疫组化图片来看,对照组和电针组海马CA3区syntaxin-1阳性表达较模型组高,对照组和电针组大鼠海马CA3区细胞质syntaxin-1表达呈空泡状,排列密集且整齐,而模型组阳性细胞数量较少,欠整齐。5.以iTRAQ技术为基础,SCX柱对样品进行液相分离及Triple TOF 5600质谱仪描绘慢性束缚应激模型组和电针干预组差异蛋白谱：(1)经过Mascot搜索引擎(version 2.3.2)在大鼠数据库IPI_rat_v3.87(共39925条序列)进行检索鉴定,本次模型组VS电针组共鉴定出33个差异蛋白,其中模型组有18个蛋白表达上调,其中包括4个不典型蛋白,模型组有15个蛋白表达下调,其中包括2个不典型蛋白。(2)通过检索Gene Ontology数据库,分析模型组VS电针组筛选出差异蛋白所参与的生物学功能,结果发现这些差异蛋白主要参与的生物过程为有机氮的生物合成代谢、离子转运、营养水平的反应以及对细胞外刺激作出的应答；主要参与的基因分子功能为离子跨膜转运、铁离子结合、多巴胺结合、儿茶酚胺结合等；其所处的细胞位置主要为细胞突起(cell projection)。(3)通过COG功能注释,结果发现除了蛋白功能未知外,COG主要集中在一般功能预测、信号转导机制、氨基酸的运输和代谢、脂质运输和代谢、能量的产生和转换、细胞周期控制和分裂、核苷酸的运输和代谢、碳水化合物的运输和代谢、转录、复制重组及修复、二级代谢产物的合成运输和分解代谢以及细胞骨架等。(4)应用Pathway显著性富集分析法确定差异蛋白参与的最主要的生化代谢途径和信号转导途径。结果发现电针对抑郁状态的调控作用主要集中在成瘾相关通路、中枢神经系统疾病相关通路、多巴胺突触以及MAPK信号转导通路等。6.验证部分选择钠依赖多巴胺转运体蛋白(Slc6a3, DAT)、酪氨酸羟化酶(Th)、微管相关蛋白delta (Tau, Mapt)、蛋白激酶C (Prkcδ)四个差异蛋白进行Western blot检验,结果发现：(1)与对照组比较,模型组大鼠海马组织DAT蛋白表达显著增加(P0.01)。电针组大鼠海马组织DAT的表达较模型组减少,但并无统计学意义(P0.05)；(2)与对照组比较,模型组大鼠海马组织Th蛋白表达有所下降,但无统计学差异(P0.05)。电针组大鼠海马组织Th的表达较模型组高,但仍然无统计学差异(P0.05)；(3)与对照组比较,模型组大鼠海马组织Krpc表达显著增加,两组有极显著差异(P0.01)。电针组大鼠海马组织Krpc的表达较模型组降低(P0.05),尽管电针组与模型组比较有所下降,但是电针组的表达较对照组高,二组具有极显著差异(P0.01)；(4)与对照组比较,模型组大鼠海马组织Tau的表达较低,二组具有显著差异(P0.05)。电针组大鼠海马组织Tau的表达较模型组显著增加(P0.05),电针组与对照组比较未见显著差异(P0.05)。结论1.行为学实验结果提示本实验慢性束缚应激结合孤养造模成功。电针在改善大鼠体质量、自主活动和探究行为方面具有绝对优势。2.电针可以改善慢性束缚应激刺激及孤养对大鼠海马神经元的损害,减少炎性介质的释放,提高突触结合蛋白的表达,从而发挥对海马神经元的保护作用,尤其是海马CA3区。3.基于iTRAQ技术对电针组和模型组大鼠海马组织蛋白进行筛查,共筛选出27个差异蛋白,对差异蛋白参与的生物学过程、基因功能和细胞定位做了初步分析,并结合HAMD七大因子团进行分析与讨论,表明抑郁症的机理研究和临床疗效评价必须从多靶点、多视角、多层面进行,并注重个性化。4.抑郁症与成瘾、中枢神经系统疾病存在共病性,电针对机体有广泛调节作用。5. DAT、Th、Krpc和Tau经western blot验证,均为电针抗抑郁的关键靶点,其中Krpc和Tau均得到与iTRAQ相一致的结果。尽管DAT和Th由于一些条件限制未能得到证实,但是基于儿茶酚胺假说,电针可能通过对Dopaminergic synapse(多巴胺突触)通路的调控作用而发挥抗抑郁效应。6. iTRAQ技术是一种稳定可靠的蛋白质组学检测方法
[Abstract]:Background and purpose in recent years, depression and drug addiction, central nervous system diseases have become the main concern of the global public health. Depression and addiction, the high CO disease of central nervous system diseases have gradually become the focus of attention. The physiological mechanism of depression is not yet clear. However, the clinical efficacy of Electroacupuncture in the treatment of depression is not clear. It has been widely recognized, which has the characteristics of curative effect, overall regulation combined with multi target treatment, and also has the attenuated and synergistic effect on drug side effects. The study of antidepressant mechanism of electroacupuncture has attracted much attention. A large number of studies have proved that electroacupuncture can be antidepressant from multiple angles, multiple targets and multiple pathways. Most of the pathology of human pathology is antidepressant. Protein plays an extremely important role in the occurrence of disease, the reaction of the body and the change of the disease. Therefore, protein is the important part of life activity. Therefore, the screening of protein markers has become a hot topic for scientists. It is necessary to explore the antidepressant mechanism of electroacupuncture at a level. The relationship between inflammatory cytokines and depressive disorder has been studied deeply. The relationship between transforming growth factor (TGF-p) and synapse binding protein (Syntaxin-1) with depression is not very common. This experiment is based on isotopic relative and absolute quantification (iTRAQ) technology. The differential protein was isolated and identified by LC-MS/MS, and the protein quantitative results were compared between the electroacupuncture group and the model group. The protein of hippocampus tissue in the rats with chronic restraint stress intervention by Electroacupuncture was screened to find the effective target of the antidepressant of electroacupuncture. Finally, the Western blot technique was used for the partial difference. Methods this study used chronic restraint stress combined with isolation to establish a rat model of depression, using open field experiment and body mass change to evaluate whether the depression model was replicated successfully, and the immunohistochemical technique was used to detect the IL-1 beta and IL-6 in the CA3 region of the hippocampus of rats. At the same time, the immuno histochemical technique was used to detect the integral light density (IOD) value of the transforming growth factor (TGF) TGF- beta and the synaptic membrane binding protein syntaxin-1 positive neurons in the CA3 region of the hippocampus of rats. The protein peptide segment of the hippocampus tissue was labeled with the iTRAQ Kit, and the strong cation exchange column (SC) was used. X) was separated, then Triple TOF 5600 mass spectrometer was used to quantify the protein polypeptide mass spectrum. After the Mascot software analysis, the differential protein mass spectrometry was depicted. After selecting the differential protein, the properties of the gene and gene products of the differential proteome of the Gene Ontology and the rat database IPI-rat_3.87 database were analyzed; through Clu. The ster of Orthologous Groups of proteins (COG) database predicts the possible functions of these proteins and classifications of their functions; determines the most important biochemical metabolic pathways and signal transduction pathways involved in the participation of differential proteins through Pathway analysis and KEGG databases. After bioinformatics analysis and literature review, the selected parts are selected. Western blot technique was used to verify the expression level of the difference protein. Results after the 1. model was completed, the horizontal movement times, vertical vertical times and body mass of the model group were significantly lower than that of the control group (P0.01). Compared with the model group, the experimental level and body mass of the electroacupuncture group increased significantly (P0.01).2. use. The IOD value of the CA3 positive neurons in the hippocampus of the hippocampus was detected by immunohistochemistry. The results showed that the expression of IL-1 beta and IL-6 in the model group was significantly increased (P0.01) compared with the control group (P0.01), and there was a significant difference between the electroacupuncture group and the model group (P0.05). The number of positive neurons in the electroacupuncture group was less than that of the model group (P0.05), and the number of the positive neurons in the electroacupuncture group was less than that of the model group. From the immunohistochemical picture, the control group was found. The number of positive cells in the hippocampal CA3 area of rats was small and ordered, and the cytoplasm coloring was shallow. The number of positive cells in the hippocampus CA3 area in the model group was significantly increased, the order of the arrangement was still clear, and the number of positive cells in the hippocampus CA3 area of the Electroacupuncture group was less than the model group, and the number of cytoplasm coloring was slightly lighter than the model group and the immunization was slightly lighter than the model group. The IOD value of TGF- beta positive neurons in the CA3 region of the hippocampus was detected by histochemical technique. The results showed that the number of positive cells in the control group and the electroacupuncture group were less and lighter, and the arrangement was neatly arranged in the control group and the electroacupuncture group, while the model group had more dense and darker positive cells, and the arrangement was slightly irregular. However, chronic restraint stress The expression of TGF- beta in the hippocampal CA3 region of rats was significantly higher than that in the control group (P0.01), but the expression was not statistically significant compared with the model group (P0.05) after the electroacupuncture intervention (P0.05).4. using immunohistochemical technique to detect the IOD value of syntaxin-1 positive neurons in the hippocampal CA3 region. The result was that after 28, the model group hippocampus was found. The expression of syntaxin-1 positive neurons in the CA3 region was significantly lower than that in the control group and the electroacupuncture group (P0.01). From the immunohistochemical picture, the positive expression of syntaxin-1 in the hippocampus CA3 region of the control group and the electroacupuncture group was higher than that in the model group. The cytoplasmic syntaxin-1 expression in the hippocampus CA3 area of the control group and the electroacupuncture group was vacuoled, and the model was dense and neatly arranged. The number of positive cells in the group was less, and the under neatly.5. was based on the iTRAQ technology. The SCX column was separated by liquid phase and the Triple TOF 5600 mass spectrometer was used to describe the differential protein spectrum of the chronic restraint stress model group and the electroacupuncture intervention group: (1) the Mascot search engine (version 2.3.2) was retrieved in the rat database IPI_rat_v3.87 (a total of 39925 sequences). A total of 33 differential proteins were identified in the model group VS electroacupuncture group, of which 18 proteins were up regulated in the model group, including 4 atypical proteins and 15 down-regulation in the model group, including 2 atypical proteins. (2) by retrieving the Gene Ontology data base, the model group VS electroacupuncture group was selected to screen the differential proteins. The biological functions of these proteins are found to be mainly involved in biosynthetic metabolism of organic nitrogen, ion transport, reaction of nutrient levels, and response to extracellular stimulation; the main functions of these proteins are ion transmembrane transport, ferric binding, dopamine binding, catecholamine binding and so on. The location of the cell is mainly cell protuberance (cell projection). (3) through the COG function annotation, it is found that in addition to the unknown protein function, COG mainly focuses on the general function prediction, the signal transduction mechanism, the transport and metabolism of amino acids, lipid transport and metabolism, the generation and conversion of energy, cell cycle control and division, and nucleotides Transport and metabolism, transport and metabolism of carbohydrates, transcription, replication, recombination and repair, synthetic transport and catabolism of two levels of metabolites, and cytoskeleton. (4) the main biochemical pathways and signal transduction pathways involved in the participation of differential proteins were determined by Pathway significant enrichment analysis. The regulation of the state is mainly concentrated in the addiction related pathway, the central nervous system disease related pathway, the dopamine synapse and the MAPK signal transduction pathway, such as.6., Slc6a3, DAT, tyrosine hydroxylase (Th), microtubule phase protein Delta (Tau, Mapt), and protein kinase C (Prkc delta) four different eggs The results of Western blot test showed that: (1) compared with the control group, the expression of DAT protein in the hippocampus of the model rats increased significantly (P0.01). The expression of DAT in the hippocampus of the electroacupuncture group was less than that in the model group, but there was no statistical significance (P0.05). (2) the expression of Th protein in the hippocampus of the model group was decreased, but the expression of Th protein in the model group was decreased, but the expression of Th protein in the model group was decreased. There was no statistical difference (P0.05). The expression of Th in hippocampus of electroacupuncture group was higher than that of model group, but there was still no statistical difference (P0.05). (3) compared with the control group, the expression of Krpc in hippocampus of model rats increased significantly (P0.01). The expression of Krpc in hippocampus of electroacupuncture group was lower than that in model group (P0.05), despite electroacupuncture (P0.05). Compared with the model group, the expression of the electroacupuncture group was higher than the control group, and the two groups had significant difference (P0.01). (4) compared with the control group, the expression of Tau in the hippocampus of the model group was lower and the two groups were significantly different (P0.05). The expression of Tau in the hippocampus group in the electroacupuncture group was significantly higher than that in the model group (P0.05), the electroacupuncture group and the opposite group were significantly higher than the model group. There was no significant difference between the two groups (P0.05). Conclusion the results of 1. behavioral experiments suggest that the experimental chronic restraint stress combined with isolated culture is successful. Electroacupuncture has absolute superiority.2. electroacupuncture in improving the body mass, autonomic activity and inquiry behavior, which can improve the damage of hippocampus neurons in rats with chronic restraint stress and isolation. The release of less inflammatory mediators, increase the expression of synaptic binding proteins, and thus play a protective role in the hippocampal neurons, especially in the hippocampal CA3 region.3. based on iTRAQ technology to screen the protein of hippocampus tissue in the electroacupuncture group and model group, screening out 27 differential proteins, the biological process, gene function and cell of differential protein. The preliminary analysis and the analysis and discussion with the seven major factor group of HAMD show that the mechanism research and clinical efficacy evaluation of depression must be from multiple targets, multiple perspectives and multilayers, and pay attention to individualized.4. depression and addiction. There is a common disease in central nervous system disease. Electroacupuncture has a wide regulating effect on the body,.5. DAT, Th, K. RPC and Tau were verified by Western blot, both of which were the key targets of electroacupuncture antidepressant, of which Krpc and Tau were all consistent with iTRAQ. Although DAT and Th were not confirmed by some conditions, electroacupuncture may be resistant to the regulation of Dopaminergic synapse (dopamine synapse) pathway based on the catecholamine hypothesis. Depression effect.6. iTRAQ technology is a stable and reliable method for proteomics detection.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R245

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