G蛋白偶联受体在催产素缓解吗啡镇痛耐受中的作用

发布时间：2018-06-05 05:22

本文选题：G蛋白偶联受体异源二聚化 + μ型阿片受体　；参考：《第二军医大学》2013年硕士论文

【摘要】：吗啡作为一种古老的、具有强大镇痛作用的药物，目前仍广泛应用于临床疼痛治疗。然而长期使用吗啡镇痛，将导致部分患者出现耐受、依赖甚至成瘾，从而大大限制了吗啡在临床上的应用。脊髓背根是伤害性刺激传入的初级中枢，吗啡在脊髓水平的镇痛作用主要通过作用于该区域的中间调节神经元，使其兴奋性降低，抑制痛觉信号上行传递，进而实现镇痛。研究表明，吗啡主要通过μ型阿片受体（MOR）和型阿片受体（DOR）发挥其药理学效应。阿片受体属于G蛋白偶联受体超家族成员，与吗啡结合将导致阿片受体构象改变，从而激活下游的G蛋白，解离的G i抑制腺苷酸环化酶的活性，，导致细胞内cAMP降低；而解离的βγ亚基直接激活膜上的G蛋白偶联的内向整流钾通道，使细胞的兴奋性降低。有研究显示慢性吗啡所引发的耐受，与吗啡不能有效地诱导阿片受体的内吞和再循环有关。促进阿片受体的内吞和再循环可有效地抑制慢性吗啡处理所诱导的耐受。 G蛋白偶联受体作为最重要的受体分子之一，对于维持机体的内稳态具有重大的意义。统计显示，有近50%的处方药物都是以G蛋白偶联受体作为靶点，其治疗作用和毒副作用不仅与其受体在组织中的分布有关，而且可能与作用的受体与其他受体的相互作用有关。越来越多的研究显示，G蛋白偶联受体（GPCRs）不仅以单体的形式发挥作用，而且还可以二聚体或多聚体形式发挥不同于单体的作用。有报道指出，型阿片受体可以与μ型阿片受体可以形成异源二聚体，并且促进吗啡诱导的μ型阿片受体的内吞。鉴于阿片受体是否内吞和再循环是吗啡诱导耐受的原因之一，那么有效地促进阿片受体内吞和再循环就可能抑制或对抗吗啡耐受的形成。我们以往的工作发现，慢性吗啡处理可以抑制大鼠视上核催产素的合成和释放，而特异性促进催产素合成和释放的锂盐可显著抑制慢性吗啡处理所导致的痛觉耐受，同时，这种效应可以被催产素受体的拮抗剂所阻断，提示催产素受体（OTR）可能参与了吗啡镇痛耐受的形成，但其作用机制目前尚不清楚。催产素受体与阿片受体同属于G蛋白偶联受体的A家族成员，但其偶联的G蛋白不同。我们以往的研究发现，催产素受体可以以还原剂敏感的多聚体形式存在，其是否可与阿片受体形成异源二聚体参与阿片受体药理学作用的调节目前尚未见报道。本研究在我们以往的工作基础上，观察阿片受体和催产素受体异源二聚化对阿片受体内吞的影响，探讨G蛋白偶联受体异源二聚化在吗啡诱导的痛觉耐受中的作用，为开发和设计新的镇痛药物提供理论和实验依据。以大鼠鞘内给药的方式建立吗啡耐受模型，观察催产素对慢性吗啡处理所诱导耐受形成的影响，结果显示吗啡连续处理至第7天，大鼠形成对吗啡的耐受，而催产素可延缓和抑制耐受的形成。免疫组化显示，大鼠脊髓背角存在着MOR与OTR的共定位；免疫共沉淀显示，脊髓中MOR与OTR存在相互作用。为了进一步确认MOR和OTR的相互作用和生理学意义，我们构建了MOR-OTR共表达转染细胞株，利用免疫共沉淀技术和双分子荧光互补（BiFC）技术，确认了两者间存在的相互作用，同时采用SNAP/CLIP染色标记的细胞影像技术，观察催产素对于吗啡诱导的MOR内吞的影响。结果表明，低剂量的催产素与吗啡联合作用时，可以有效地促进MOR的内吞，而吗啡和催产素单独处理对MOR的内吞没有影响。为了研究MOR和OTR相互作用可能的功能区域，我们表达和纯化了MOR的四个功能区段的GST融合蛋白，利用GST-Pull Down研究介导MOR与OTR相互作用的区段。结果显示，μ型阿片受体第三个胞内环可能参与了其与催产素受体之间的相互作用。本实验所得结论如下： 1、鞘内注射催产素，可以显著提高吗啡的镇痛效率，延迟耐受的出现。 2、在大鼠脊髓存在着μ型阿片受体与催产素受体的相互作用。并且在脊髓背角，首次观察到了μ型阿片受体与催产素受体的共定位。 3、在体外共表达体系中，μ型阿片受体与催产素受体有相互作用关系；低剂量的催产素可以有效促进吗啡诱导的μ型阿片受体的内吞。 4、 μ型阿片受体的第三个胞内环，参与了催产素受体与其的相互作用。
[Abstract]:Morphine, as an old, powerful analgesic drug, is still widely used in clinical pain treatment. However, the long-term use of morphine for analgesia will lead to tolerance, dependence and addiction in some patients, which greatly restricts the clinical application of morphine.
The dorsal root of the spinal cord is the primary center of the nociceptive stimulation. The analgesic effect of morphine at the spinal level is mainly mediated by the intermediate regulating neurons in the region, which reduces the excitability, inhibits the transmission of the pain signal and then realizes the analgesia. The study shows that morphine mainly passes through the MOR and DOR. The opioid receptor belongs to the member of the G protein coupling receptor superfamily. The binding of the opioid receptor to the morphine will lead to the change of the opioid receptor conformation, which activates the downstream G protein, and the dissociated G I inhibits the activity of adenylate cyclase and leads to the decrease in the intracellular cAMP, while the dissociated beta subunit directly activates the inward rectifier potassium on the membrane coupled with the G protein. The channel reduces the excitability of the cell.
Studies have shown that the tolerance induced by chronic morphine is related to the failure of morphine to effectively induce endocytosis and recirculation of opioid receptors. Promoting endocytosis and recirculation of opioid receptors can effectively inhibit the tolerance induced by chronic morphine treatment.
G protein coupled receptor, one of the most important receptor molecules, is of great significance for maintaining the homeostasis of the body. Statistics show that nearly 50% of the prescription drugs are targeted by G protein coupled receptors. Their therapeutic effects and side effects are not only related to the distribution of their receptors in the tissues, but also may be associated with their receptors and their receptors. The interaction of his receptor is related. More and more studies have shown that G protein coupled receptors (GPCRs) not only play a role in the form of monomers, but can also play a different form of monomers in the form of two polymers or polymers.
Opioid receptors can form heteromeric two dimers with opioid receptors and promote morphine induced endocytosis of the opioid receptor.
In view of whether endocytosis and recirculation of opioid receptors are one of the causes of morphine induced tolerance, the effective promotion of opioid endocytosis and recirculation may inhibit or antagonize the formation of morphine tolerance.
Our previous work has found that chronic morphine treatment inhibits the synthesis and release of oxytocin in the optic nucleus of the rat, and the lithium salts that specifically promote the synthesis and release of oxytocin can significantly inhibit the pain tolerance induced by chronic morphine treatment, and this effect can be blocked by oxytocin receptor antagonists, suggesting that the oxytocin receptor (OT R) may be involved in the formation of morphine tolerance, but its mechanism is not yet clear. Oxytocin receptor and opioid receptor are members of the A family of the G protein coupled receptor, but their coupling G proteins are different. Our previous study found that oxytocin receptor can exist in the form of a reductant sensitive polymer, and it can be used with opiate. The regulation of opioid receptor pharmacological effects by the formation of heterologous two dimers has not been reported.
On the basis of our previous work, we observe the effect of opioid receptor and oxytocin receptor heteromerization on opioid receptor endocytosis, and explore the role of heterologous dimerization of G protein coupled receptors in morphine induced pain tolerance, and provide theoretical and experimental basis for the development and design of new analgesic drugs.
The effect of oxytocin on the induced tolerance induced by chronic morphine treatment was observed in the form of morphine tolerance in the rat's sheath. The results showed that morphine was treated for seventh days and the rats were tolerant to morphine, while oxytocin could delay and inhibit the formation of tolerance. Immunohistochemistry showed that MOR and OTR existed in the dorsal horn of the spinal cord of rats. Co localization; immunoprecipitation showed that there was interaction between MOR and OTR in the spinal cord. In order to further confirm the interaction and physiological significance of MOR and OTR, we constructed a MOR-OTR co expression transfected cell line, and used immunoprecipitation technique and double molecular fluorescence complementation (BiFC) technology to confirm the interaction between them and use S. The effect of oxytocin on morphine induced MOR endocytosis was observed by NAP/CLIP staining. The results showed that the combined action of low dose oxytocin and morphine could effectively promote endocytosis of MOR, while morphine and oxytocin alone did not respond to the endocytosis of MOR. In order to study the possible function of the interaction of MOR and OTR In the region, we express and purify the GST fusion protein of four functional segments of MOR, and use GST-Pull Down to study the section of the interaction between MOR and OTR. The results show that the third intracellular rings of the UA opioid receptor may participate in their interaction with the oxytocin receptor.
The results obtained in this experiment are as follows:
1, intrathecal oxytocin can significantly improve the analgesic efficiency of morphine and delay the occurrence of tolerance.
2, the interaction between the opioid receptor and oxytocin receptor is present in the spinal cord of the rat, and the co localization of the UA opioid receptor and oxytocin receptor is observed for the first time in the dorsal horn of the spinal cord.
3, in the coexpression system in vitro, there is a interaction between the opioid receptor and oxytocin receptor, and the low dose of oxytocin can effectively promote the endocytosis of morphine induced opioid receptor.
4, the third intracellular rings of the opioid receptor are involved in the interaction between oxytocin receptor and its receptor.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.61

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