發(fā)布時(shí)間：2018-06-24 10:37

  本文選題：抑郁癥 + 色氨酸耗竭　； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景 抑郁癥是一種持久的心境低落狀態(tài)，其核心癥狀為情緒低落、興趣缺乏、樂(lè)趣喪失、并伴有焦慮、自責(zé)自罪、自殺行為及睡眠障礙等。抑郁癥已經(jīng)成為當(dāng)今世界危害人類(lèi)健康最大的疾病之一，，其終身患病率為5%~17%。抑郁癥的致病機(jī)制目前仍然不明確，且缺乏客觀的診斷指標(biāo)。選擇性5-HT再攝取抑制劑（SSRIs）類(lèi)抗抑郁藥在臨床上取得的療效提示，抑郁癥的發(fā)病與5-HT系統(tǒng)功能密切相關(guān)。色氨酸耗竭試驗(yàn)通過(guò)耗竭體外攝入的色氨酸，影響外周及中樞系統(tǒng)中5-HT的含量及功能，是研究5-HT系統(tǒng)改變的有效方法。建立色氨酸耗竭模型，并對(duì)模型動(dòng)物的腦組織進(jìn)行蛋白質(zhì)組學(xué)分析的研究還未見(jiàn)報(bào)道。 目的 本課題將基礎(chǔ)研究與臨床醫(yī)療相結(jié)合，遵循轉(zhuǎn)化醫(yī)學(xué)的研究思路，通過(guò)建立大鼠快速色氨酸耗竭模型，分析5-羥色胺能系統(tǒng)與動(dòng)物抑郁樣行為發(fā)生的聯(lián)系，并運(yùn)用比較蛋白質(zhì)組學(xué)結(jié)合質(zhì)譜技術(shù)對(duì)模型動(dòng)物的海馬組織進(jìn)行分離鑒定，旨在從機(jī)體蛋白質(zhì)層面探究疾病的發(fā)病機(jī)制，為臨床抑郁癥診斷做一有益探索。 方法 本課題共分為三部分：第一部分，大鼠快速色氨酸耗竭模型的建立與評(píng)價(jià)；第二部分，大鼠快速色氨酸耗竭模型組織的比較蛋白質(zhì)組學(xué)研究。 雄性Sprague-Dawley大鼠，體重300g~350g，行為學(xué)篩選后隨機(jī)分為三組：快速色氨酸耗竭模型組（TRP-組），含色氨酸的膠原蛋白對(duì)照組（TRP+組），鹽水對(duì)照組（Saline組）。適應(yīng)性灌胃10天后進(jìn)行常規(guī)灌胃造模，采用糖水消耗實(shí)驗(yàn)和敞箱實(shí)驗(yàn)對(duì)動(dòng)物進(jìn)行行為學(xué)檢測(cè)，運(yùn)用高效液相色譜技術(shù)檢測(cè)分析造模后動(dòng)物血漿中的氨基酸含量。 選擇與抑郁癥發(fā)病相關(guān)的海馬區(qū)進(jìn)行單個(gè)樣本的比較蛋白質(zhì)組學(xué)分析。三氯乙酸-丙酮沉淀法提取組織蛋白，結(jié)合雙向電泳分離技術(shù)，ImageMaster2D Platinum6.0軟件對(duì)2-DE圖像進(jìn)行分析，MALDI-TOF-MS質(zhì)譜鑒定差異蛋白，所得質(zhì)譜數(shù)據(jù)在NCBInr數(shù)據(jù)庫(kù)中檢索蛋白相關(guān)信息，分析蛋白功能。 結(jié)果 造模后，各組動(dòng)物的飲水量均有所下降，其中TRP-a組的糖水偏嗜度顯著低于Saline組和TRP+組（P均0.01），與其自身的基線數(shù)據(jù)也有顯著差異（P0.05）。TRP-a組動(dòng)物活動(dòng)的總路程顯著低于Saline組和TRP+組（P均0.05），與其自身的基線數(shù)據(jù)也有顯著差異（P0.05）。動(dòng)物造模后氨基酸檢測(cè)結(jié)果顯示，與Saline組和TRP+組相比，快速色氨酸耗竭模型組（TRP-組）均表現(xiàn)出色氨酸含量明顯下降，且具有顯著性差異（P0.001）；與Saline組和TRP+組相比，觀察指標(biāo)TRP/∑LNAA在TRP-組也表現(xiàn)出顯著性差異（P0.001）。 ImageMaster2D Platinum6.0軟件對(duì)2-DE圖像進(jìn)行分析，各組間兩兩比較，共分析出88個(gè)差異表達(dá)的蛋白質(zhì)（%Vol1，P0.05），并對(duì)其中14個(gè)差異蛋白進(jìn)行了質(zhì)譜鑒定。差異蛋白基本分為以下6類(lèi)：（1）細(xì)胞骨架相關(guān)蛋白：如Septin-11基因編碼的蛋白；（2）神經(jīng)發(fā)育相關(guān)蛋白：如二氫嘧啶酶相關(guān)3、髓鞘堿性蛋白；（3）信號(hào)轉(zhuǎn)導(dǎo)相關(guān)蛋白：如生長(zhǎng)因子受體結(jié)合蛋白2、Stathmin；（4）能量代謝相關(guān)蛋白：如NADH脫氫酶、H+-ATPase；（5）轉(zhuǎn)錄和調(diào)控相關(guān)因子：如Sirtuin2、泛素結(jié)合酶E2N、受翻譯調(diào)節(jié)的腫瘤蛋白；（6）氧化代謝相關(guān)蛋白：如氨基�；�-1A、谷胱甘肽轉(zhuǎn)移酶Mu1、3-磷酸甘油脫氫酶。 結(jié)論 高效液相色譜檢測(cè)血漿氨基酸數(shù)據(jù)表明，動(dòng)物模型成功耗竭了外周血中的色氨酸，且由TRP/∑LNAA指標(biāo)可知，由外周血漿進(jìn)入中樞神經(jīng)系統(tǒng)的色氨酸量也明顯降低，并推測(cè)由色氨酸生成的5-HT量也會(huì)明顯降低。通過(guò)動(dòng)物行為學(xué)評(píng)價(jià)，快速色氨酸耗竭試驗(yàn)?zāi)苣M人類(lèi)5-TH能系統(tǒng)改變所導(dǎo)致的抑郁情緒、快感缺失、活動(dòng)減少等情緒。模型操作簡(jiǎn)單、安全、可逆，具備可行性。 檢索文獻(xiàn)，分析差異蛋白功能，發(fā)現(xiàn)其對(duì)抑郁癥的相關(guān)研究有重要意義。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R749.4

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