發(fā)布時間：2018-03-26 08:14

  本文選題：嗎啡　切入點：μ阿片受體　出處：《河北醫(yī)科大學》2016年碩士論文

【摘要】：嗎啡作為臨床上強效鎮(zhèn)痛藥物已經有百年的歷史,但是它的耐受性和潛在依賴性不僅限制了其應用,而且會導致阿片成癮。截至2015年底我國登記在冊吸毒人員234.5萬人,濫用阿片類毒品人員占40%以上,而實際吸毒人員預計將達到1000萬人以上。目前,嗎啡鎮(zhèn)痛的機制研究較為清楚:嗎啡與μ阿片受體結合,導致G蛋白亞基分解,產生Gαi和Gβ亞基,抑制AC(腺苷酸環(huán)化酶),抑制c AMP(環(huán)磷酸腺苷),引起細胞內鈣離子通道關閉,鉀離子、鈉離子通道開放,達到鎮(zhèn)痛效果。然而嗎啡鎮(zhèn)痛產生失敏和耐受的機制研究尚未完全清楚,目前認為可能與μ阿片受體功能下調、μ阿片受體磷酸化、G蛋白偶聯(lián)受體激酶、PKC(蛋白激酶C)的調節(jié)等有關。嗎啡導致的耐受會經歷短期失敏、長期失敏、耐受這樣一個過程,并且每個過程都與μ阿片受體的磷酸化有密切關系。膽囊收縮素(cholecystokinin,CCK)作為一種典型的腦腸肽,以神經遞質和神經調質的形式發(fā)揮著重要作用,并且八肽膽囊收縮素(cholecystokinin octapeptide,CCK-8)是目前已知作用最強的內源性“抗阿片肽”,通過“負反饋”機制調節(jié)阿片耐受和依賴過程。我們的研究結果顯示,CCK-8可以通過激活CCK2受體可抑制阿片受體的結合力,發(fā)揮“抗阿片”作用,CCK2受體拮抗劑可以抑制嗎啡依賴的形成;而大劑量CCK-8通過刺激CCK1受體反而可以使內源性阿片物質的釋放增加,CCK1受體拮抗劑可以部分翻轉外源性CCK-8對嗎啡依賴的抑制作用。所以推斷,CCK受體的不同亞型以及受體的不同敏感狀態(tài)可能對嗎啡依賴均具有不同的調控作用。然而,目前由于缺少CCK-1受體的商品化配體,CCK-1受體與CCK-2受體通常同時存在于組織、細胞中,缺少對兩種受體單獨研究的實驗模型。基于以上研究背景,本實驗將以HEK-293(人腎上皮細胞系)細中單獨穩(wěn)定過表達μ阿片受體(以下簡稱μ細胞)、穩(wěn)定過表達μ阿片受體和CCK-1受體(以下簡稱K1細胞)、穩(wěn)定過表達μ阿片受體和CCK-2受體(以下簡稱K2細胞)三種細胞作為實驗模型,研究μ阿片受體磷酸化與受體短期失敏的關系,觀察CCK-8通過不同受體亞型對μ阿片受體磷酸化的調控作用,并初步探究CCK-8通過不同亞型發(fā)揮作用的機制。第一部分:CCK-8對嗎啡引起的μ阿片受體持續(xù)磷酸化的調控作用目的:證明嗎啡能引起μ阿片受體持續(xù)磷酸化,從而導致μ阿片受體短期失敏,并觀察CCK-8通過不同受體亞型對其調控作用。方法:1在μ細胞上給予100μM嗎啡、10μM DAMGO急性作用30μin,隨后撤除刺激0.5h、1h、2h、3h、4h、5h、6h,在各個時間點用western blot法檢測磷酸化μ阿片受體表達變化;2在K1、K2細胞上給予1μM CCK-8預處理12h,給予嗎啡100μM急性作用30μin,撤除刺激3h,用western blot法檢測磷酸化μ阿片受體表達變化,觀察CCK-8對嗎啡引起的持續(xù)磷酸化的調控,并觀察是通過哪種受體亞型發(fā)揮作用;3取正常μ細胞(正常組),上述10μM DAMGO刺激30μin后撤除刺激3h的μ細胞(以下簡稱D3組),100μM嗎啡刺激30μin后撤除刺激3h的μ細胞(以下簡稱M3組),1μMCCK-8預處理12h后100μM嗎啡刺激30μin再撤除刺激3h的k2細胞(以下簡稱M+C 3組),再次給予10μM嗎啡刺激5μin,用RT-PCR的方法,檢測G蛋白偶聯(lián)受體兩個亞基Gαi和Gβ的μ RNA水平的表達,證明持續(xù)磷酸化與受體失敏之間的聯(lián)系,觀察CCK-8對受體失敏的調控作用。結果:1嗎啡對μ阿片受體磷酸化的作用嗎啡急性作用會引起持續(xù)的磷酸化(P0.05),撤除嗎啡刺激3h后磷酸化水平并未下降;DAMGO引起的μ阿片受體磷酸化隨著刺激的撤除,磷酸化水平迅速降低(P0.01),到3h組降低至對照組水平(P0.001)。2 CCK-8對μ阿片受體持續(xù)磷酸化的調控作用2.1過表達CCK1受體細胞中,未能觀察到嗎啡急性作用下μ阿片受體磷酸化現(xiàn)象(P0.05);2.2過表達CCK2受體細胞中,CCK-8預處理細胞12h后,不僅能抑制嗎啡引起的μ阿片受體磷酸化(P0.01),同時加速撤除刺激后μ阿片受體去磷酸化過程(P0.001)。3 CCK-8對嗎啡引起的μ阿片受體短期失敏的調控作用D3組(DAMGO刺激30μin后撤除刺激3h的μ細胞)、M3組(嗎啡啡刺激30μin后撤除刺激3h的μ細胞)、M+C3組(CCK-8預處理12h后嗎啡刺激30μin再撤除刺激3h的k2細胞)再次給予10μM嗎啡刺激5μin,D3組Gαi和Gβ的μ RNA水平與對照組并無差異(P0.05),M3組Gαi和Gβ的μ RNA的表達較D3組顯著降低(P0.001),M+C3組Gαi和Gβ的μ RNA水平的表達較M3組明顯升高(P0.01)。提示嗎啡急性作用下,μ阿片受體失敏程度與其磷酸化水平成正比,CCK-8能通過CCK-2受體加速μ阿片受體去磷酸化水平緩解嗎啡急性作用引起的受體短期失敏。第二部分:CCK-8調控嗎啡引起的μ阿片受體持續(xù)磷酸化的機制探討目的:1觀察CCK-8對μ阿片受體磷酸化的調控作用是否與PKC(蛋白激酶C)有關;2觀察在嗎啡作用下,μ阿片受體是否會與CCK1受體或CCK2受體形成功能性二聚體,共同發(fā)揮調節(jié)作用。方法:1 在μ細胞上分別給予100μM嗎啡,10μM DAMGO刺激30μin,western blot方法檢測PKC表達的變化;2 1μM CCK-8預處理K1、K2細胞,再加入100μM嗎啡刺激30μin,western blot方法檢測PKC表達的變化,觀察CCK-8通過不同受體亞型作用時PKC含量的變化;3 在μ細胞上給予100μM嗎啡刺激30μin,撤除刺激1h、2h、3h、4h、5h,在K2細胞上1μM CCK-8預處理12h,100μM嗎啡刺激30μin,撤除刺激1h、2h、3h、4h、5h,在各個時間點用western blot法檢測PKC表達的變化,探究PKC含量變化過程與磷酸化μ阿片受體的變化過程的關系;4 我室前期構建的μ、K1、K2細胞中,μ阿片受體連接Histag標簽抗體,CCK1受體連接V5標簽抗體,CCK2受體連接Flag標簽抗體。K1、K2細胞給予100μM嗎啡作用30μin(嗎啡急性作用)和10μM嗎啡作用12h(嗎啡慢性作用),用CO-IP方法檢測Histag、V5和Flag抗體,從而證明μ阿片受體和CCK1受體或CCK2受體之間是否存在相互作用。結果:1 PKC在嗎啡引起的μ阿片受體磷酸化中的調節(jié)作用1.1嗎啡急性作用會引起PKC含量顯著升高(P0.001),而DAMGO急性作用下PKC含量與control組比無差異(P0.05);1.2過表達CCK1受體細胞中,嗎啡急性作用不會引起PKC含量上升(P0.05),但CCK-8預處理組PKC含量均升高(P0.001);1.3過表達CCK2受體細胞中,CCK-8預處理細胞能降低嗎啡引起的PKC含量增高(P0.001);1.4單獨嗎啡急性作用下,撤除嗎啡刺激后,PKC含量下降具有延遲性,3h組PKC含量下降才有統(tǒng)計學意義(P0.01),5h組下降至control組水平;過表達CCK-2受體的細胞經過CCK-8預處理后,給予嗎啡作用后撤除刺激,1h組PKC含量下降有統(tǒng)計學意義(P0.01),4h組下降至control組水平。上述結果提示:CCK-8能通過CCK-2受體抑制嗎啡急性作用引起的PKC含量升高,并能加速撤除嗎啡刺激后PKC的下降過程,與其加速μ阿片受體去磷酸化的過程一致,提示PKC參與CCK-8調節(jié)嗎啡引起μ片受體的磷酸化過程。2μ阿片受體與CCK1受體、CCK2受體相互作用免疫共沉淀實驗中發(fā)現(xiàn),μ阿片受體能與CCK1受體形成二聚體,共同發(fā)揮作用,同時并未檢測到μ阿片受體與CCK2受體形成二聚體。上述結果提示:CCK1受體能與μ阿片受體形成功能性二聚體,從而翻轉嗎啡引起的μ阿片受體磷酸化及PKC含量升高。結論:1嗎啡引起的μ阿片受體持續(xù)磷酸化會導致受體短期失敏,CCK-8能通過CCK-2受體加速μ阿片受體去磷酸化過程,從而緩解嗎啡所致的受體短期失敏;2嗎啡使PKC蛋白含量升高,CCK-8能通過CCK2受體加速PKC含量下降的過程,與其加速μ阿片受體去磷酸化過程一致,提示PKC參與CCK-8調節(jié)嗎啡引起的μ阿片受體持續(xù)磷酸化過程;3 CCK1受體與μ阿片受體可以形成二聚體,從而翻轉嗎啡引起的μ阿片受體磷酸化及PKC含量升高的現(xiàn)象。
[Abstract]:Morphine as clinically potent analgesic drugs have been a hundred years of history, but the tolerance and potential dependence of it not only limits its application, and will lead to opioid addiction. As of the end of 2015 China's 2 million 345 thousand registered drug addicts, abuse of opioid drugs staff accounted for more than 40%, while the actual drug users is expected to to reach more than 10 million people. At present, research on the mechanism of morphine analgesia is clear: morphine combined with opioid receptors, leading to G protein subunit G decomposition, alpha I and G beta subunit AC (inhibition of adenylate cyclase (AMP), the inhibition of C acid, adenosine cyclophosphate) induced by calcium ion channel cell in closing, potassium ion, sodium ion channels open, to achieve the analgesic effect of morphine analgesia. However the mechanism of desensitization and tolerance are not fully understood, is considered to be down regulated with mu opioid receptors, opioid receptor phosphorylation, G protein coupled receptor kinase , PKC (protein kinase C) the regulation and so on. Morphine induced tolerance will experience short-term desensitization, long-term desensitization, tolerance of such a process, and there is a close relationship between phosphorylation and opioid receptor. Each process of cholecystokinin (cholecystokinin, CCK) brain gut peptides are a kind of typical. The neurotransmitter and neuromodulator form plays an important role, and the eight octapeptide (cholecystokinin octapeptide CCK-8) is the "strongest known role of endogenous anti opioid peptides", through the "negative feedback regulation mechanism of opioid tolerance and dependence. Our results show that CCK-8 can activate CCK2 through binding force the receptor can inhibit opioid receptors, play anti opioid effect, CCK2 receptor antagonists can inhibit the formation of morphine dependence; while large dose of CCK-8 by stimulating CCK1 receptors but can make endogenous opioid substances The increased release of CCK1 receptor antagonists can inhibit partially reversed exogenous CCK-8 on morphine dependence. So that different sensitive CCK receptor isoforms and receptors may have different regulatory effects on morphine dependence. However, due to the commercialization of ligands lacking the CCK-1 receptor, CCK-1 receptor and CCK-2 receptor usually at the same time in tissue cells, the lack of experimental studies on two kinds of receptor model separately. Based on the above research background, this experiment will be HEK-293 (human renal epithelial cell line) fine separately in the over expression of mu opioid receptor (hereinafter referred to as mu, cells) over expression of mu opioid receptor and CCK-1 receptor (hereinafter referred to as K1) cells, over expression of mu opioid receptor and CCK-2 receptor (K2 cells) three cells as an experimental model of mu opioid receptor phosphorylation and desensitization of opioid receptor short-term relationship, observation CCK -8 through different receptor subtypes regulate opioid receptor phosphorylation, and explore CCK-8 mechanisms through different subtypes. The first part: the objective regulation of opioid receptor CCK-8 on morphine induced sustained phosphorylation that morphine can cause mu opioid receptor phosphorylation and sustained. Lead to short-term desensitization of opioid receptor, and observe the CCK-8 through different receptor subtypes in the regulation. Methods: 1 mu in cells given 100 M morphine 10 M DAMGO acute effect of 30 in, 0.5h 1H, then removes the stimulation, 2h, 3h, 4h, 5h, 6h, expression detection of phosphorylation of opioid receptor by Western blot method at each time point; 2 in K1, given 1 M CCK-8 pretreatment 12h K2 cells, morphine 100 M acute effect of 30 in, the removal of 3H by Western stimulation, blot method was used to detect the phosphorylation of mu opioid receptor expression, continuous observation of phosphorus CCK-8 on morphine induced 閰稿寲鐨勮皟鎺，

本文編號：1667125