本文選題：創(chuàng)傷后應(yīng)激障礙　切入點：單次長時程應(yīng)激　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:本課題采用單次長時程應(yīng)激(single prolonged stress,SPS)作為創(chuàng)傷后應(yīng)激障礙模型,綜合行為學(xué)、化學(xué)遺傳學(xué)方法、免疫熒光等技術(shù)手段觀察杏仁核不同的神經(jīng)元集群在大鼠PTSD樣癥狀中的作用及相關(guān)神經(jīng)生物學(xué)機制,旨在為開發(fā)新的PTSD臨床治療手段和方案提供理論依據(jù)。方法:首先,采用SPS模型進(jìn)行造模,將SD大鼠隨機分成未造模對照組,SPS模型造模后1d組,造模后14d組,利用曠場實驗、高架十字迷宮實驗檢測大鼠PTSD的焦慮樣行為改變,利用免疫熒光技術(shù)檢測杏仁核的c-fos蛋白表達(dá)變化,并用c-fos來表征腦區(qū)的激活,探究杏仁核的激活在大鼠PTSD樣行為發(fā)生中的作用。其次,采用化學(xué)遺傳學(xué)方法,即DREADDs技術(shù)(designer receptors exclusively activated by designer drugs)來抑制杏仁核腦區(qū),檢測其是否影響PTSD的發(fā)生。通過腦區(qū)注射腺相關(guān)病毒,將改造的人M4毒蕈堿受體hM4Di表達(dá)在大鼠杏仁核CeA腦區(qū),并通過腹腔注射hM4Di的配體氯氮平-N-氧化物(clozapine-N-oxides,CNO)來實現(xiàn)杏仁核的抑制。采用SPS模型進(jìn)行造模,利用曠場實驗、高架十字迷宮檢測PTSD的焦慮樣行為學(xué)改變,驗證杏仁核參與PTSD樣癥狀的發(fā)生。最后,利用免疫熒光技術(shù)檢測BLA和CeA的c-fos表達(dá)變化,并用來表征其腦區(qū)激活;同時應(yīng)用谷氨酸能神經(jīng)元的分子標(biāo)簽CaMKII和GABA能神經(jīng)元的分子標(biāo)簽GAD67和c-fos共標(biāo)來表征不同的神經(jīng)元集群的激活情況,探究相關(guān)機制。結(jié)果:1.同未經(jīng)SPS造模的對照組大鼠相比較,SPS造模后14d的大鼠在曠場中央?yún)^(qū)和高架十字的開臂停留時間均顯著的縮短(P0.05),SPS造模后1d組沒有明顯差異,表明SPS可誘導(dǎo)大鼠產(chǎn)生延遲性的焦慮樣行為。c-fos免疫熒光結(jié)果顯示在CeA腦區(qū),同未經(jīng)SPS造模的對照組大鼠相比,造模14d后c-fos的表達(dá)含量增高(P0.05),但是與造模后1d的大鼠相比,沒有明顯差異。而BLA腦區(qū)神經(jīng)元集群的激活模式未發(fā)現(xiàn)任何顯著性改變。2.利用DREADD技術(shù)抑制杏仁核CeA腦區(qū),同其他未干預(yù)組相比,SPS造模后14d大鼠在曠場中央?yún)^(qū)和高架十字的開臂停留時間均顯著的增加(P0.05),表現(xiàn)出抗焦慮。3.大鼠在SPS模型后,在免疫熒光技術(shù)檢測試驗中,GAD67+c-fos陽性/c-fos陽性的比值顯示在CeA腦區(qū),同未造模動物相比,造模14天后GAD67+c-fos陽性/c-fos陽性的比值明顯增高[F(2,16)=13.06,P0.001,],有統(tǒng)計學(xué)意義,但是造模后第1天沒有表現(xiàn)明顯差異,而BLA腦區(qū)GABA能神經(jīng)元集群的激活模式未發(fā)現(xiàn)任何顯著性改變。CaMKⅡ+c-fos陽性/c-fos陽性的比值顯示在CeA腦區(qū)和BLA腦區(qū),谷氨酸能神經(jīng)元集群的激活模式都未發(fā)現(xiàn)任何顯著性改變。結(jié)論:1.大鼠PTSD樣行為的發(fā)生同時伴隨有中央杏仁核的激活,這可能參與了PTSD發(fā)生。2.大鼠中央杏仁核CeA腦區(qū)的抑制,出現(xiàn)抗焦慮作用,中央杏仁核參與PTSD的發(fā)生。3.中央杏仁核的GABA能神經(jīng)元的激活比例的失調(diào)很可能參與了PTSD樣行為的發(fā)生。其因果聯(lián)系需要進(jìn)一步的化學(xué)遺傳學(xué)進(jìn)一步證明。
[Abstract]:Objective: this topic by a single minister Cheng Yingji (single prolonged stress, SPS) as a model of posttraumatic stress disorder, comprehensive behavioral methods, chemical genetics, immunofluorescence technique was used to observe the different clusters of amygdala neurons in rat PTSD like symptoms in use and bioneurological mechanism, aiming to provide a theoretical basis for the development of the new PTSD clinical treatment methods and programs. Methods: firstly, SPS model is used to model, SD rats were randomly divided into non model control group, SPS model group 1D, model group 14d, using the open field test, the change of anxiety like behavior in the elevated plus maze test in rats PTSD to detect the expression of amygdala, c-fos protein by immunofluorescence technique, and using c-fos to characterize the activation of brain regions, the activation of the amygdala in rats on PTSD like behavior. Secondly, using chemical genetics Methods, namely DREADDs Technology (designer receptors exclusively activated by designer drugs) to suppress the amygdala, detect whether or not influence the occurrence of PTSD. Through the brain injection of adeno-associated virus, the transformation of the human M4 muscarinic receptor hM4Di expression in the amygdala of CeA rat brain, and the ligand clozapine were intraperitoneally injected with hM4Di (-N- oxide clozapine-N-oxides, CNO) to achieve the amygdala inhibition by SPS model. Modeling, using the open field test, the change of anxiety like behavior in the elevated plus maze test PTSD, test PTSD like symptoms in the amygdala. Finally, the BLA expression of CeA and c-fos by immunofluorescence technique, and used to characterize the brain activation at the same time; application of glutamatergic neurons and molecular labels CaMKII and GABA neurons GAD67 and c-fos labeled molecular tags to neuron clusters of different characterization of the bowel Live, to explore the related mechanism. Results: 1. without rats compared with the control of SPS, SPS after the model of 14d rats were significantly reduced in the open arm time, open field and elevated central cross (P0.05), there was no significant difference in SPS model group 1D, showed that SPS rats can induce delayed anxiety like behavior of.C-fos immunofluorescence results showed CeA in brain regions, without SPS model control group rats compared with model 14d expression increased the content of c-fos (P0.05), but with the mice in 1D rats compared to no significant difference. BLA clusters of neurons in brain activation patterns did not find any significant change in.2. using DREADD technology to inhibit amygdala CeA brain regions, compared with other non intervention group, after SPS 14d rats in the open arm time central open field and elevated plus were significantly increased (P0.05),.3. exhibited anti anxiety rats in SPS In the model, immunofluorescence assay test, the ratio of GAD67+c-fos positive /c-fos positive display in the CeA brain regions, compared with an animal model, modeling the ratio of GAD67+c-fos positive /c-fos positive after 14 days were significantly increased [F (2,16) =13.06, P0.001, have statistical significance, but not first days after modeling the obvious difference, while the GABA BLA brain neurons cluster activation patterns did not find any significant change in the ratio of.CaMK II +c-fos positive /c-fos positive CeA and BLA displayed in the brain areas of the brain, glutamate can activate the pattern of neuronal clusters are found no significant changes. Conclusion: 1. rat PTSD like behavior at the same time accompanied by the activation of the central nucleus of the amygdala, which may be involved in the suppression of PTSD central amygdala CeA cortex of.2. rats, have anti anxiety effects, the central nucleus of the amygdala is involved in the development of PTSD.3. GABA central amygdala neurons The maladjustment of the activation ratio of the element is likely to be involved in the occurrence of PTSD like behavior. The causal link requires further evidence of chemical genetics.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Bart Borghans;Judith R Homberg;;Animal models for posttraumatic stress disorder: An overview of what is used in research[J];World Journal of Psychiatry;2015年04期

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本文編號：1632303