本文選題：抑郁 + CUMS　； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:觀察慢性應(yīng)激大鼠的抑郁行為與皮層神經(jīng)元COX2表達變化的相關(guān)性,并從皮層COX2-EPs-BDNF通路初步探討抑郁癥發(fā)病機制。方法:1.大鼠抑郁模型的建立與皮層中AA-COX2通路改變:雄性SD大鼠20只,隨機分為正常組(n=10)和模型組(n=10),正常組大籠飼養(yǎng),每籠5只,自由攝水進食且不給予任何刺激;模型組孤養(yǎng)并給予慢性不可預(yù)見性溫和刺激(chronic unpredictable mild stress,CUMS),連續(xù)42d,造模完成后進行行為學(xué)測試。2.COX2干預(yù)對慢性應(yīng)激大鼠抑郁行為的影響:(1)雄性SD大鼠50只,隨機分為5組(n=10),即正常對照組、模型組、陽性藥物對照組、美洛昔康1 mg?kg-1組和美洛昔康3 mg?kg-1組,正常組大鼠處理同第一部分,其余四組大鼠孤養(yǎng)并給予42d CUMS,給藥組大鼠分別灌胃給予舍曲林(5 mg?kg-1)、美洛昔康(1、3 mg?kg-1),模型組與正常組給予等體積0.5%CMC-Na,每天一次,連續(xù)21d,給藥完成后進行行為學(xué)測試。(2)另取雄性SD大鼠40只,并將其隨機分為3組,即空載病毒(control)組(n=10)大籠飼養(yǎng),自由攝水進食且不給予任何刺激;COX2過表達慢病毒注射+CUMS組(n=10)和空載病毒+CUMS組(n=20)。大鼠施行側(cè)腦室定位注射導(dǎo)管埋管,從術(shù)后第5天開始給予cums,并分別在術(shù)后第5d,12d,19d進行腦室內(nèi)慢病毒注射,每只15μl/次。給予cums42d后進行行為學(xué)測試,空載病毒+cums組大鼠隨機分為兩組,即cox2rnai(n=10)和cums組(n=10),分別腦室內(nèi)注射cox2rnai慢病毒和空載病毒,每7天一次,共3次,每只15μl/次,第3次注射后第7d進行行為學(xué)測試。在以上分組和干預(yù)的基礎(chǔ)上采用糖水實驗、曠場實驗和強迫游泳實驗檢測大鼠行為學(xué)變化,以蘇木精-伊紅染色觀察大鼠皮層神經(jīng)元形態(tài)變化;elisa和生化酶學(xué)法測定大鼠皮層組織pge2、tnf-α、il-1β、camp、mda含量和sod活性;免疫組化檢測皮層bdnf蛋白表達情況;rt-pcr檢測大鼠皮層cox2mrna表達;westernblot檢測cox2、ep2、ep3、pkaⅡαreg、creb、p-creb、bdnf蛋白含量。結(jié)果:1.與正常組相比,cums大鼠糖水偏好率下降(p0.01),曠場實驗水平(p0.01)和垂直得分(p0.01)減少,強迫游泳靜止不動時間增加(p0.01);皮層神經(jīng)元出現(xiàn)明顯核固縮;皮層pge2、tnf-α、il-1β、mda含量增加(p0.01),而sod活性下降(p0.01);皮層cox2mrna和蛋白的表達(p0.01)均增加。2.與單獨給予cums的空載病毒大鼠相比,在給予大鼠cums的同時側(cè)腦室注射過表達cox2慢病毒,能進一步降低大鼠糖水偏好率,明顯減少大鼠曠場水平和垂直得分(p0.05),增加強迫游泳不動時間(p0.05);增加皮層pge2、tnf-α、il-1β、mda含量(p0.01或P0.05)和降低SOD活性(P0.05),減少c AMP含量(P0.05),顯著增加COX2 mRNA(P0.05)表達的同時,COX2、EP2和EP3蛋白表達有上升趨勢,PKAⅡαreg蛋白表達顯著減少(P0.05),而p-CREB、BDNF蛋白表達有下降趨勢。當(dāng)給予美洛昔康抑制COX2活性或側(cè)腦室注射COX2 RNAi慢病毒,則能明顯提高抑郁大鼠糖水偏好率(P0.05),增加大鼠曠場實驗水平和垂直得分(P0.01),減少強迫游泳不動時間(P0.05);顯著減少皮層PGE2、TNF-α、IL-1β、MDA含量(P0.01)和增加SOD活性(P0.01);明顯增加皮層c AMP含量(P0.01)和PKAⅡαreg、p-CREB、BDNF蛋白表達(P0.01或P0.05),下調(diào)COX2 mRNA(P0.01)和蛋白(P0.05)以及EP2(P0.01)、EP3(P0.05)蛋白的表達。結(jié)論:CUMS致抑郁大鼠皮層中AA-COX2通路明顯激活。抑制COX2活性或降低其在大腦中的表達可明顯改善抑郁大鼠的抑郁行為;而COX2過表達可增加大鼠對CUMS的易感性,加重抑郁癥狀。皮層AA-COX2通路激活參與抑郁癥發(fā)病機制可能涉及下游產(chǎn)物PGE2激動EP2、EP3受體,直接影響c AMP/PKA-CREB-BDNF通路的活化程度,從而改變皮層神經(jīng)元可塑性。
[Abstract]:Objective: To observe the correlation between the depressive behavior of chronic stress rats and the changes of COX2 expression in cortical neurons, and to explore the pathogenesis of depression from the cortical COX2-EPs-BDNF pathway. Methods: the establishment of the depression model in 1. rats and the change of the AA-COX2 pathway in the cortex: 20 male SD rats were randomly divided into normal group (n=10) and model group (n=10), normal and normal. The group was fed with 5 cages, free of water intake and no stimulation. The model group was isolated and given chronic unpredictability and mild stimulation (chronic unpredictable mild stress, CUMS) and continuous 42d. After the completion of the model, the behavioral test was carried out by.2.COX2 intervention on the depressive behavior of chronic irritable rats: (1) 50 male SD rats were randomly selected. Divided into 5 groups (n=10), namely normal control group, model group, positive drug control group, meloxicam 1 mg? Kg-1 group and meloxicam 3 mg? Kg-1 group, normal group rats were treated with the first part, the other four groups of rats were isolated and given 42d CUMS, and the rats in the drug group were given Seculin (5 mg? Kg-1), meloxicam (1,3 mg? Kg-1), model group and normal Group given equal volume 0.5%CMC-Na, once a day, continuous 21d, after the completion of the drug test. (2) another 40 male SD rats were taken, and they were randomly divided into 3 groups, namely, the empty virus (control) group (n=10) large cage, free intake of water intake and no prickly stimulation; COX2 overexpression lentivirus +CUMS group (n=10) and the empty virus +CUMS group ( N=20). The rats performed the lateral ventricle with intraventricular catheter buried tube, and were given cums from fifth days after the operation. The intraventricular lentivirus was injected at 5D, 12D, and 19d after the operation, each 15 l/ times. After cums42d, the behavior test was carried out. The empty virus +cums group rats were randomly divided into two groups, cox2rnai (n=10) and cums group (n=10), intraventricular injection respectively. Cox2rnai lentivirus and no-load virus were shot, once every 7 days, 3 times, each 15 mu l/ times, and after third injection, the behavior test was tested at 7d. On the basis of the above group and intervention, the behavior changes of rats were detected by the sugar water experiment, the open field experiment and the forced swimming test, and the morphological changes of the cortical neurons in the rats were observed with hematoxylin eosin staining; e Determination of PGE2, tnf- alpha, IL-1 beta, camp, MDA content and SOD activity in rat cortical tissue by Lisa and biochemical enzymology; immunohistochemistry to detect the expression of BDNF protein in cortex; RT-PCR detection of cortical cox2mrna expression in rats; Westernblot detection COX2. The rate decreased (P0.01), the level of open field experiment (P0.01) and the vertical score (P0.01) decreased, the resting time of forced swimming increased (P0.01), the cortical neurons had obvious nuclear pyknosis, the cortex PGE2, tnf- a, IL-1 beta, MDA content increased (P0.01), and SOD activity decreased (P0.01), and the expression of cortical cox2mrna and protein increased and was given alone. Compared with the no-load virus, the injection of COX2 lentivirus in the lateral ventricle of rat cums could further reduce the sugar water preference rate, reduce the level and vertical score (P0.05) of rats and increase the time of forced swimming (P0.05), and increase the PGE2, tnf- alpha, IL-1 beta, MDA content (P0.01 or P0.05) and SOD activity (P0.) (P0.) (P0.) and SOD activity (P0.). 05), decrease the content of C AMP (P0.05), significantly increase the expression of COX2 mRNA (P0.05), while COX2, EP2 and EP3 protein expression has a rising trend, PKA II alpha reg protein expression significantly decreased (P0.05). Rats' sugar water preference rate (P0.05) increased rats' open field experiment level and vertical score (P0.01), reduced forced swimming time (P0.05), decreased cortical PGE2, TNF- alpha, IL-1 beta, MDA content (P0.01) and increased SOD activity (P0.01). MRNA (P0.01) and protein (P0.05) and the expression of EP2 (P0.01), EP3 (P0.05) protein. Conclusion: the AA-COX2 pathway in the cortex of depressive rats is obviously activated by CUMS. Inhibiting the activity of COX2 or decreasing its expression in the brain can obviously improve the depressive behavior of the depressed rats, and COX2 overexpression can increase the susceptibility to CUMS and aggravate the depressive symptoms. The activation of the layer AA-COX2 pathway involved in the pathogenesis of depression may involve the downstream product PGE2 excited EP2, the EP3 receptor, which directly affects the activation of the C AMP/PKA-CREB-BDNF pathway, and thus changes the plasticity of cortical neurons.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R749.4

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