發(fā)布時間：2018-04-27 05:26

  本文選題：差異蛋白 + 電針　； 參考：《北京中醫(yī)藥大學》2015年博士論文

【摘要】：背景與目的近年來,抑郁癥與藥物成癮、中樞神經(jīng)系統(tǒng)疾病成為全球公共健康關注的主要問題。抑郁癥與成癮、中樞神經(jīng)系統(tǒng)疾病的高共病性也逐漸成為關注的熱點。目前抑郁癥的病生理機制尚不清楚。但是電針治療抑郁癥的臨床療效已經(jīng)得到了廣泛的認可,具有療效確切,整體調(diào)節(jié)結合多靶點治療的特點,同時對藥物的毒副作用也有減毒增效作用。電針抗抑郁機理研究已經(jīng)備受關注,大量研究證實電針能從多角度、多靶點、多途徑起到抗抑郁作用。人類絕大多數(shù)病理生理過程都是由蛋白質(zhì)參與完成的,在疾病的發(fā)生、機體的反應以及疾病轉(zhuǎn)歸的每一個過程,蛋白質(zhì)都發(fā)揮著極其重要的作用,因此,蛋白質(zhì)作為生命活動的重要組成部分,蛋白標志物的篩選就成為科學家們關注的熱點。因此從蛋白質(zhì)水平探討電針抗抑郁機制是十分必要的。關于致炎性細胞因子與抑郁癥發(fā)病的關系已有較深入的研究,而轉(zhuǎn)化生長因子(TGF-p)及突觸結合蛋白(Syntaxin-1)與抑郁癥的關系研究尚不多見。本實驗基于同位素相對與絕對定量(iTRAQ)技術,聯(lián)合多維液相色譜技術與串聯(lián)質(zhì)譜技術(LC-MS/MS)分離和鑒定差異蛋白,比較電針組與模型組海馬組織蛋白定量結果,對電針所干預的慢性束縛應激大鼠的海馬組織蛋白進行初篩,尋找電針抗抑郁的有效靶點,最后,選用Western blot技術對部分差異蛋白進行驗證。方法本研究應用慢性束縛應激結合孤養(yǎng)建立抑郁大鼠模型,采用曠場實驗、體質(zhì)量變化情況來評價抑郁模型是否復制成功,并應用免疫組化技術對大鼠海馬組織CA3區(qū)炎性細胞因子IL-1β、IL-6進行檢測,確認大鼠是否出現(xiàn)免疫炎癥反應；同時,采用免疫組化技術對大鼠海馬組織CA3區(qū)不同于其他炎性因子的轉(zhuǎn)化生長因子TGF-β、突觸膜結合蛋白syntaxin-1陽性神經(jīng)元的積分光密度(IOD)值進行檢測；采用iTRAQ試劑盒標記海馬組織蛋白肽段,經(jīng)強陽離子交換柱(SCX)分離,之后使用Triple TOF 5600質(zhì)譜儀對蛋白質(zhì)多肽質(zhì)譜圖進行定量鑒定,經(jīng)過Mascot軟件分析后,描繪差異蛋白質(zhì)譜。挑選出差異蛋白后,登陸Gene Ontology與大鼠數(shù)據(jù)庫IPI-rat_3.87數(shù)據(jù)庫進行差異蛋白質(zhì)的基因和基因產(chǎn)物的屬性分析；通過Cluster of Orthologous Groups of proteins (COG)數(shù)據(jù)庫預測這些蛋白質(zhì)可能的功能并對其功能做分類統(tǒng)計；通過Pathway分析與KEGG數(shù)據(jù)庫確定差異蛋白參與的最主要的生化代謝途徑和信號轉(zhuǎn)導途徑。經(jīng)過生物信息學分析及文獻查閱后,挑選出部分差異蛋白,應用Western blot技術驗證其表達水平。結果1.造模完成后,模型組大鼠曠場實驗的水平運動次數(shù)、垂直豎立次數(shù)、體質(zhì)量均顯著低于對照組(P0.01)說明造模成功；與模型組相比,電針組曠場實驗水平及體質(zhì)量明顯升高(P0.01)。2.采用免疫組化技術檢測海馬組織CA3區(qū)IL-1β和IL-6陽性神經(jīng)元的IOD值。結果發(fā)現(xiàn)：模型組IL-1β、IL-6的表達與對照組相比明顯增多(p0.01)；電針組與模型組比較有顯著差異(p0.05),電針組陽性神經(jīng)元數(shù)量少于模型組。從免疫組化圖片來看,對照組大鼠海馬CA3區(qū)陽性細胞數(shù)量較少且排列有序,胞漿著色較淺；模型組大鼠海馬CA3區(qū)陽性細胞數(shù)量明顯增多,排列整齊程度尚可,著色深；電針組大鼠海馬CA3區(qū)陽性細胞數(shù)量較模型組少,且排列較有序,胞漿著色較模型組略淺。3.采用免疫組化技術檢測海馬組織CA3區(qū)TGF-β陽性神經(jīng)元的IOD值。結果發(fā)現(xiàn)：從免疫組化圖片來看對照組和電針組陽性細胞數(shù)量較少且著色較淺,排列較整齊。而模型組則分布有較多密集、著色較深的陽性細胞,且排列略不整齊。然而,慢性束縛應激結合孤養(yǎng)能夠引起大鼠海馬組織CA3區(qū)TGF-β表達較對照組明顯升高(p0.01),但是電針干預后其表達與模型組相比卻沒有統(tǒng)計學意義(p0.05)。4.采用免疫組化技術檢測海馬組織CA3區(qū)syntaxin-1陽性神經(jīng)元的IOD值。結果發(fā)現(xiàn)：經(jīng)過28后,模型組海馬CA3區(qū)syntaxin-1陽性神經(jīng)元的表達與對照組和電針組比較明顯下降(p0.01)。從免疫組化圖片來看,對照組和電針組海馬CA3區(qū)syntaxin-1陽性表達較模型組高,對照組和電針組大鼠海馬CA3區(qū)細胞質(zhì)syntaxin-1表達呈空泡狀,排列密集且整齊,而模型組陽性細胞數(shù)量較少,欠整齊。5.以iTRAQ技術為基礎,SCX柱對樣品進行液相分離及Triple TOF 5600質(zhì)譜儀描繪慢性束縛應激模型組和電針干預組差異蛋白譜：(1)經(jīng)過Mascot搜索引擎(version 2.3.2)在大鼠數(shù)據(jù)庫IPI_rat_v3.87(共39925條序列)進行檢索鑒定,本次模型組VS電針組共鑒定出33個差異蛋白,其中模型組有18個蛋白表達上調(diào),其中包括4個不典型蛋白,模型組有15個蛋白表達下調(diào),其中包括2個不典型蛋白。(2)通過檢索Gene Ontology數(shù)據(jù)庫,分析模型組VS電針組篩選出差異蛋白所參與的生物學功能,結果發(fā)現(xiàn)這些差異蛋白主要參與的生物過程為有機氮的生物合成代謝、離子轉(zhuǎn)運、營養(yǎng)水平的反應以及對細胞外刺激作出的應答；主要參與的基因分子功能為離子跨膜轉(zhuǎn)運、鐵離子結合、多巴胺結合、兒茶酚胺結合等；其所處的細胞位置主要為細胞突起(cell projection)。(3)通過COG功能注釋,結果發(fā)現(xiàn)除了蛋白功能未知外,COG主要集中在一般功能預測、信號轉(zhuǎn)導機制、氨基酸的運輸和代謝、脂質(zhì)運輸和代謝、能量的產(chǎn)生和轉(zhuǎn)換、細胞周期控制和分裂、核苷酸的運輸和代謝、碳水化合物的運輸和代謝、轉(zhuǎn)錄、復制重組及修復、二級代謝產(chǎn)物的合成運輸和分解代謝以及細胞骨架等。(4)應用Pathway顯著性富集分析法確定差異蛋白參與的最主要的生化代謝途徑和信號轉(zhuǎn)導途徑。結果發(fā)現(xiàn)電針對抑郁狀態(tài)的調(diào)控作用主要集中在成癮相關通路、中樞神經(jīng)系統(tǒng)疾病相關通路、多巴胺突觸以及MAPK信號轉(zhuǎn)導通路等。6.驗證部分選擇鈉依賴多巴胺轉(zhuǎn)運體蛋白(Slc6a3, DAT)、酪氨酸羥化酶(Th)、微管相關蛋白delta (Tau, Mapt)、蛋白激酶C (Prkcδ)四個差異蛋白進行Western blot檢驗,結果發(fā)現(xiàn)：(1)與對照組比較,模型組大鼠海馬組織DAT蛋白表達顯著增加(P0.01)。電針組大鼠海馬組織DAT的表達較模型組減少,但并無統(tǒng)計學意義(P0.05)；(2)與對照組比較,模型組大鼠海馬組織Th蛋白表達有所下降,但無統(tǒng)計學差異(P0.05)。電針組大鼠海馬組織Th的表達較模型組高,但仍然無統(tǒng)計學差異(P0.05)；(3)與對照組比較,模型組大鼠海馬組織Krpc表達顯著增加,兩組有極顯著差異(P0.01)。電針組大鼠海馬組織Krpc的表達較模型組降低(P0.05),盡管電針組與模型組比較有所下降,但是電針組的表達較對照組高,二組具有極顯著差異(P0.01)；(4)與對照組比較,模型組大鼠海馬組織Tau的表達較低,二組具有顯著差異(P0.05)。電針組大鼠海馬組織Tau的表達較模型組顯著增加(P0.05),電針組與對照組比較未見顯著差異(P0.05)。結論1.行為學實驗結果提示本實驗慢性束縛應激結合孤養(yǎng)造模成功。電針在改善大鼠體質(zhì)量、自主活動和探究行為方面具有絕對優(yōu)勢。2.電針可以改善慢性束縛應激刺激及孤養(yǎng)對大鼠海馬神經(jīng)元的損害,減少炎性介質(zhì)的釋放,提高突觸結合蛋白的表達,從而發(fā)揮對海馬神經(jīng)元的保護作用,尤其是海馬CA3區(qū)。3.基于iTRAQ技術對電針組和模型組大鼠海馬組織蛋白進行篩查,共篩選出27個差異蛋白,對差異蛋白參與的生物學過程、基因功能和細胞定位做了初步分析,并結合HAMD七大因子團進行分析與討論,表明抑郁癥的機理研究和臨床療效評價必須從多靶點、多視角、多層面進行,并注重個性化。4.抑郁癥與成癮、中樞神經(jīng)系統(tǒng)疾病存在共病性,電針對機體有廣泛調(diào)節(jié)作用。5. DAT、Th、Krpc和Tau經(jīng)western blot驗證,均為電針抗抑郁的關鍵靶點,其中Krpc和Tau均得到與iTRAQ相一致的結果。盡管DAT和Th由于一些條件限制未能得到證實,但是基于兒茶酚胺假說,電針可能通過對Dopaminergic synapse(多巴胺突觸)通路的調(diào)控作用而發(fā)揮抗抑郁效應。6. iTRAQ技術是一種穩(wěn)定可靠的蛋白質(zhì)組學檢測方法
[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.

【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R245

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