本文選題：嗎啡依賴 + 黑質(zhì)（SN）　； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：嗎啡依賴是指以失去控制能力、強(qiáng)迫性連續(xù)用藥為特征的慢性復(fù)發(fā)性腦疾病，不僅對成癮者身心健康造成嚴(yán)重的損害，而且對社會造成嚴(yán)重的不良影響，已經(jīng)成為社會普遍關(guān)注的焦點(diǎn)問題。目前，國內(nèi)外對嗎啡依賴的研究主要限于機(jī)能及功能代謝方面，病理形態(tài)學(xué)方面的研究尚不夠詳細(xì)。大量研究表明中腦多巴胺能神經(jīng)系統(tǒng)幾乎參與了所有依賴性藥物的獎賞效應(yīng)，被認(rèn)為是獎賞效應(yīng)的最后通路。因此中腦多巴胺能神經(jīng)系統(tǒng)是藥物成癮最關(guān)鍵的腦區(qū)之一。 目前的研究表明嗎啡對全身各系統(tǒng)存在不同程度的損害，其對心、肺的病理損害，加上嗎啡對中樞神經(jīng)系統(tǒng)的直接作用以及對呼吸中樞的抑制等，可引起大腦急、慢性缺血缺氧，進(jìn)而引起神經(jīng)細(xì)胞、神經(jīng)纖維發(fā)生相應(yīng)的病理性改變。但病理形態(tài)學(xué)方面的研究未見詳細(xì)報道，因此本研究采用多巴胺能神經(jīng)細(xì)胞特異性標(biāo)記物酪氨酸羥化酶(tyrosine hydroxylase,TH)，進(jìn)行免疫組織化學(xué)染色，從病理形態(tài)學(xué)角度系統(tǒng)觀察不同時程嗎啡依賴對中腦黑質(zhì)(Substantia nigra, SN)及腹側(cè)被蓋區(qū)(ventral tegmental area,VTA)多巴胺能神經(jīng)細(xì)胞的影響。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)，較長時程嗎啡依賴大鼠SN及VTA多巴胺能神經(jīng)細(xì)胞數(shù)目明顯減少，為了探明不同時程嗎啡依賴導(dǎo)致多巴胺能神經(jīng)細(xì)胞的減少是由于嗎啡的神經(jīng)毒性引發(fā)了細(xì)胞的變性壞死還是引發(fā)了凋亡，本實(shí)驗(yàn)先后從整體水平和細(xì)胞水平兩個層面進(jìn)行了研究。首先通過建立不同時程嗎啡依賴大鼠模型，觀察不同時程嗎啡依賴對中腦SN及VTA多巴胺能神經(jīng)細(xì)胞的影響，之后采用多巴胺能性的MN9D細(xì)胞系，從細(xì)胞水平系統(tǒng)觀察了不同時程嗎啡刺激對多巴胺能神經(jīng)細(xì)胞的影響，為進(jìn)一步探索與形態(tài)改變相關(guān)聯(lián)的功能性變化打下基礎(chǔ)。 方法： 1本實(shí)驗(yàn)采用Wistar大鼠，體重為(300±20)g的健康雌性大鼠48只，分為空白對照組2周組、嗎啡依賴2周組，空白對照組4周組、嗎啡依賴4周組，，空白對照組6周組和嗎啡依賴6周組。嗎啡依賴3組大鼠按逐日遞增原則，背部皮下注射鹽酸嗎啡，每天二次(8:00，20:00)，連續(xù)5天。劑量分別為10mg·Kg~(-1)，20mg·Kg~(-1)，30mg·Kg~(-1)，40mg·Kg~(-1)，50mg·kg-1。每次注射前均用75%酒精嚴(yán)格消毒，一次性注射器注射。5天后確認(rèn)嗎啡依賴形成后，嗎啡依賴2周組、4周組、6周組分別給予30mg·Kg~(-1)鹽酸嗎啡背部皮下注射，一天兩次(8:00，20:00)，分別連續(xù)注射至2周、4周、6周，建立三個不同時段的嗎啡依賴大鼠模型�？瞻讓φ�2周組、4周組、6周組分別注射等劑量的生理鹽水，分別連續(xù)注射至2周、4周、6周。模型建立后，腦組織按照腦立體定位圖譜標(biāo)準(zhǔn)部位切取后經(jīng)脫水、透明、包埋、連續(xù)切片，免疫組織化學(xué)及免疫熒光染色觀察SN及VTA多巴胺能神經(jīng)細(xì)胞TH的表達(dá)情況；Fluoro-Jade B染色以及原位末端標(biāo)記法(TUNEL)檢測嗎啡依賴不同時程中腦多巴胺能神經(jīng)細(xì)胞變性壞死及凋亡情況。 2采用多巴胺能性的MN9D細(xì)胞系，利用免疫熒光對MN9D細(xì)胞系上的、、三種阿片受體進(jìn)行檢測。嗎啡作用48h、24h、12h后，F(xiàn)luoro-JadeB染色觀察不同時程嗎啡刺激對多巴胺能性MN9D細(xì)胞的影響。 利用SPSS16.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)，所有數(shù)據(jù)均用均數(shù)±標(biāo)準(zhǔn)差表示，采用兩個獨(dú)立樣本t檢驗(yàn)，P0.05為有顯著性差異。 結(jié)果： 1.免疫組化結(jié)果顯示較長時程嗎啡依賴TH陽性細(xì)胞體積減小，細(xì)胞胞漿著色加深，TH陽性細(xì)胞數(shù)目減少，與正常對照組比有顯著性差異。 2. Fluoro-Jade B染色結(jié)果顯示較長時程嗎啡依賴SN及VTA多巴胺能神經(jīng)細(xì)胞發(fā)生了變性壞死。 3.原位末端標(biāo)記法(TUNEL)結(jié)果顯示未見明顯凋亡細(xì)胞。 4.細(xì)胞Fluoro-Jade B染色結(jié)果顯示較長時程嗎啡刺激后MN9D細(xì)胞發(fā)生了變性壞死。 結(jié)論： 較長時程嗎啡依賴SN及VTA多巴胺能神經(jīng)細(xì)胞明顯減少，其減少主要是由于長期的嗎啡刺激所產(chǎn)生的神經(jīng)毒性作用導(dǎo)致多巴胺能神經(jīng)細(xì)胞發(fā)生了變性壞死引起的。
[Abstract]:Objective: morphine dependence is a chronic recurrent brain disease characterized by loss of control and compulsive continuous medication. It not only causes serious damage to the physical and mental health of the addicts, but also has serious adverse effects on society. It has become a focus of attention in the society. At present, the study of morphine dependence at home and abroad is limited to the main limit. A large number of studies have shown that the mesencephalon dopaminergic nervous system is almost involved in the reward effect of all dependent drugs and is considered to be the final pathway of the reward effect. Therefore, the mesencephalic dopaminergic system is one of the most critical brain areas for drug addiction.
The present study shows that morphine has different degrees of damage to the systemic system, the pathological damage to the heart, the lung, the direct effect of morphine on the central nervous system and the inhibition of the respiratory center, which can cause acute and chronic ischemic anoxia in the brain, and then cause the neuropathic and pathological changes of the nerve fibers, but the pathology of the nerve fibers. The morphological study was not reported in detail. Therefore, the tyrosine hydroxylase (TH), a dopaminergic neuron specific marker, was used for immunohistochemical staining. From the pathomorphological point of view, Cheng Mafei dependent on the mesencephalic substantia nigra (Substantia nigra, SN) and the ventral tegmental area (VE). The effects of ntral tegmental area, VTA) on dopaminergic neurons. Experimental results showed that the number of neurons of SN and VTA dopaminergic neurons in long term morphine dependent rats decreased significantly, and the decrease of dopaminergic neurons caused by morphine dependence in different periods was due to the neurotoxicity of endorphins that caused degeneration and necrosis of the cells. Apoptosis was initiated, and the experiment was carried out from two levels of overall level and cell level. First, the effects of different time history morphine dependence on SN and VTA dopaminergic neurons of the middle brain were observed by different time history morphine dependent rats. Then the dopaminergic MN9D cell line was observed from the cell level system. The effects of morphine stimulation at different time intervals on dopaminergic neurons are the basis for further exploration of functional changes associated with morphological changes.
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