CCK-8对吗啡引起的μ阿片受体短期失敏的调控作用及机制探讨

发布时间：2018-03-26 08:14

  本文选题：吗啡　切入点：μ阿片受体　出处：《河北医科大学》2016年硕士论文

【摘要】：吗啡作为临床上强效镇痛药物已经有百年的历史,但是它的耐受性和潜在依赖性不仅限制了其应用,而且会导致阿片成瘾。截至2015年底我国登记在册吸毒人员234.5万人,滥用阿片类毒品人员占40%以上,而实际吸毒人员预计将达到1000万人以上。目前,吗啡镇痛的机制研究较为清楚:吗啡与μ阿片受体结合,导致G蛋白亚基分解,产生Gαi和Gβ亚基,抑制AC(腺苷酸环化酶),抑制c AMP(环磷酸腺苷),引起细胞内钙离子通道关闭,钾离子、钠离子通道开放,达到镇痛效果。然而吗啡镇痛产生失敏和耐受的机制研究尚未完全清楚,目前认为可能与μ阿片受体功能下调、μ阿片受体磷酸化、G蛋白偶联受体激酶、PKC(蛋白激酶C)的调节等有关。吗啡导致的耐受会经历短期失敏、长期失敏、耐受这样一个过程,并且每个过程都与μ阿片受体的磷酸化有密切关系。胆囊收缩素(cholecystokinin,CCK)作为一种典型的脑肠肽,以神经递质和神经调质的形式发挥着重要作用,并且八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)是目前已知作用最强的内源性“抗阿片肽”,通过“负反馈”机制调节阿片耐受和依赖过程。我们的研究结果显示,CCK-8可以通过激活CCK2受体可抑制阿片受体的结合力,发挥“抗阿片”作用,CCK2受体拮抗剂可以抑制吗啡依赖的形成;而大剂量CCK-8通过刺激CCK1受体反而可以使内源性阿片物质的释放增加,CCK1受体拮抗剂可以部分翻转外源性CCK-8对吗啡依赖的抑制作用。所以推断,CCK受体的不同亚型以及受体的不同敏感状态可能对吗啡依赖均具有不同的调控作用。然而,目前由于缺少CCK-1受体的商品化配体,CCK-1受体与CCK-2受体通常同时存在于组织、细胞中,缺少对两种受体单独研究的实验模型。基于以上研究背景,本实验将以HEK-293(人肾上皮细胞系)细中单独稳定过表达μ阿片受体(以下简称μ细胞)、稳定过表达μ阿片受体和CCK-1受体(以下简称K1细胞)、稳定过表达μ阿片受体和CCK-2受体(以下简称K2细胞)三种细胞作为实验模型,研究μ阿片受体磷酸化与受体短期失敏的关系,观察CCK-8通过不同受体亚型对μ阿片受体磷酸化的调控作用,并初步探究CCK-8通过不同亚型发挥作用的机制。第一部分:CCK-8对吗啡引起的μ阿片受体持续磷酸化的调控作用目的:证明吗啡能引起μ阿片受体持续磷酸化,从而导致μ阿片受体短期失敏,并观察CCK-8通过不同受体亚型对其调控作用。方法:1在μ细胞上给予100μM吗啡、10μM DAMGO急性作用30μin,随后撤除刺激0.5h、1h、2h、3h、4h、5h、6h,在各个时间点用western blot法检测磷酸化μ阿片受体表达变化;2在K1、K2细胞上给予1μM CCK-8预处理12h,给予吗啡100μM急性作用30μin,撤除刺激3h,用western blot法检测磷酸化μ阿片受体表达变化,观察CCK-8对吗啡引起的持续磷酸化的调控,并观察是通过哪种受体亚型发挥作用;3取正常μ细胞(正常组),上述10μM DAMGO刺激30μin后撤除刺激3h的μ细胞(以下简称D3组),100μM吗啡刺激30μin后撤除刺激3h的μ细胞(以下简称M3组),1μMCCK-8预处理12h后100μM吗啡刺激30μin再撤除刺激3h的k2细胞(以下简称M+C 3组),再次给予10μM吗啡刺激5μin,用RT-PCR的方法,检测G蛋白偶联受体两个亚基Gαi和Gβ的μ RNA水平的表达,证明持续磷酸化与受体失敏之间的联系,观察CCK-8对受体失敏的调控作用。结果:1吗啡对μ阿片受体磷酸化的作用吗啡急性作用会引起持续的磷酸化(P0.05),撤除吗啡刺激3h后磷酸化水平并未下降;DAMGO引起的μ阿片受体磷酸化随着刺激的撤除,磷酸化水平迅速降低(P0.01),到3h组降低至对照组水平(P0.001)。2 CCK-8对μ阿片受体持续磷酸化的调控作用2.1过表达CCK1受体细胞中,未能观察到吗啡急性作用下μ阿片受体磷酸化现象(P0.05);2.2过表达CCK2受体细胞中,CCK-8预处理细胞12h后,不仅能抑制吗啡引起的μ阿片受体磷酸化(P0.01),同时加速撤除刺激后μ阿片受体去磷酸化过程(P0.001)。3 CCK-8对吗啡引起的μ阿片受体短期失敏的调控作用D3组(DAMGO刺激30μin后撤除刺激3h的μ细胞)、M3组(吗啡啡刺激30μin后撤除刺激3h的μ细胞)、M+C3组(CCK-8预处理12h后吗啡刺激30μin再撤除刺激3h的k2细胞)再次给予10μM吗啡刺激5μin,D3组Gαi和Gβ的μ RNA水平与对照组并无差异(P0.05),M3组Gαi和Gβ的μ RNA的表达较D3组显著降低(P0.001),M+C3组Gαi和Gβ的μ RNA水平的表达较M3组明显升高(P0.01)。提示吗啡急性作用下,μ阿片受体失敏程度与其磷酸化水平成正比,CCK-8能通过CCK-2受体加速μ阿片受体去磷酸化水平缓解吗啡急性作用引起的受体短期失敏。第二部分:CCK-8调控吗啡引起的μ阿片受体持续磷酸化的机制探讨目的:1观察CCK-8对μ阿片受体磷酸化的调控作用是否与PKC(蛋白激酶C)有关;2观察在吗啡作用下,μ阿片受体是否会与CCK1受体或CCK2受体形成功能性二聚体,共同发挥调节作用。方法:1 在μ细胞上分别给予100μM吗啡,10μM DAMGO刺激30μin,western blot方法检测PKC表达的变化;2 1μM CCK-8预处理K1、K2细胞,再加入100μM吗啡刺激30μin,western blot方法检测PKC表达的变化,观察CCK-8通过不同受体亚型作用时PKC含量的变化;3 在μ细胞上给予100μM吗啡刺激30μin,撤除刺激1h、2h、3h、4h、5h,在K2细胞上1μM CCK-8预处理12h,100μM吗啡刺激30μin,撤除刺激1h、2h、3h、4h、5h,在各个时间点用western blot法检测PKC表达的变化,探究PKC含量变化过程与磷酸化μ阿片受体的变化过程的关系;4 我室前期构建的μ、K1、K2细胞中,μ阿片受体连接Histag标签抗体,CCK1受体连接V5标签抗体,CCK2受体连接Flag标签抗体。K1、K2细胞给予100μM吗啡作用30μin(吗啡急性作用)和10μM吗啡作用12h(吗啡慢性作用),用CO-IP方法检测Histag、V5和Flag抗体,从而证明μ阿片受体和CCK1受体或CCK2受体之间是否存在相互作用。结果:1 PKC在吗啡引起的μ阿片受体磷酸化中的调节作用1.1吗啡急性作用会引起PKC含量显著升高(P0.001),而DAMGO急性作用下PKC含量与control组比无差异(P0.05);1.2过表达CCK1受体细胞中,吗啡急性作用不会引起PKC含量上升(P0.05),但CCK-8预处理组PKC含量均升高(P0.001);1.3过表达CCK2受体细胞中,CCK-8预处理细胞能降低吗啡引起的PKC含量增高(P0.001);1.4单独吗啡急性作用下,撤除吗啡刺激后,PKC含量下降具有延迟性,3h组PKC含量下降才有统计学意义(P0.01),5h组下降至control组水平;过表达CCK-2受体的细胞经过CCK-8预处理后,给予吗啡作用后撤除刺激,1h组PKC含量下降有统计学意义(P0.01),4h组下降至control组水平。上述结果提示:CCK-8能通过CCK-2受体抑制吗啡急性作用引起的PKC含量升高,并能加速撤除吗啡刺激后PKC的下降过程,与其加速μ阿片受体去磷酸化的过程一致,提示PKC参与CCK-8调节吗啡引起μ片受体的磷酸化过程。2μ阿片受体与CCK1受体、CCK2受体相互作用免疫共沉淀实验中发现,μ阿片受体能与CCK1受体形成二聚体,共同发挥作用,同时并未检测到μ阿片受体与CCK2受体形成二聚体。上述结果提示:CCK1受体能与μ阿片受体形成功能性二聚体,从而翻转吗啡引起的μ阿片受体磷酸化及PKC含量升高。结论:1吗啡引起的μ阿片受体持续磷酸化会导致受体短期失敏,CCK-8能通过CCK-2受体加速μ阿片受体去磷酸化过程,从而缓解吗啡所致的受体短期失敏;2吗啡使PKC蛋白含量升高,CCK-8能通过CCK2受体加速PKC含量下降的过程,与其加速μ阿片受体去磷酸化过程一致,提示PKC参与CCK-8调节吗啡引起的μ阿片受体持续磷酸化过程;3 CCK1受体与μ阿片受体可以形成二聚体,从而翻转吗啡引起的μ阿片受体磷酸化及PKC含量升高的现象。
[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，

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