發(fā)布時(shí)間：2018-06-05 05:22

  本文選題：G蛋白偶聯(lián)受體異源二聚化 + μ型阿片受體��； 參考：《第二軍醫(yī)大學(xué)》2013年碩士論文

【摘要】：嗎啡作為一種古老的、具有強(qiáng)大鎮(zhèn)痛作用的藥物，目前仍廣泛應(yīng)用于臨床疼痛治療。然而長(zhǎng)期使用嗎啡鎮(zhèn)痛，將導(dǎo)致部分患者出現(xiàn)耐受、依賴甚至成癮，從而大大限制了嗎啡在臨床上的應(yīng)用。 脊髓背根是傷害性刺激傳入的初級(jí)中樞，嗎啡在脊髓水平的鎮(zhèn)痛作用主要通過作用于該區(qū)域的中間調(diào)節(jié)神經(jīng)元，使其興奮性降低，抑制痛覺信號(hào)上行傳遞，進(jìn)而實(shí)現(xiàn)鎮(zhèn)痛。研究表明，嗎啡主要通過μ型阿片受體（MOR）和型阿片受體（DOR）發(fā)揮其藥理學(xué)效應(yīng)。阿片受體屬于G蛋白偶聯(lián)受體超家族成員，與嗎啡結(jié)合將導(dǎo)致阿片受體構(gòu)象改變，從而激活下游的G蛋白，解離的G i抑制腺苷酸環(huán)化酶的活性，，導(dǎo)致細(xì)胞內(nèi)cAMP降低；而解離的βγ亞基直接激活膜上的G蛋白偶聯(lián)的內(nèi)向整流鉀通道，使細(xì)胞的興奮性降低。 有研究顯示慢性嗎啡所引發(fā)的耐受，與嗎啡不能有效地誘導(dǎo)阿片受體的內(nèi)吞和再循環(huán)有關(guān)。促進(jìn)阿片受體的內(nèi)吞和再循環(huán)可有效地抑制慢性嗎啡處理所誘導(dǎo)的耐受。 G蛋白偶聯(lián)受體作為最重要的受體分子之一，對(duì)于維持機(jī)體的內(nèi)穩(wěn)態(tài)具有重大的意義。統(tǒng)計(jì)顯示，有近50%的處方藥物都是以G蛋白偶聯(lián)受體作為靶點(diǎn)，其治療作用和毒副作用不僅與其受體在組織中的分布有關(guān)，而且可能與作用的受體與其他受體的相互作用有關(guān)。越來越多的研究顯示，G蛋白偶聯(lián)受體（GPCRs）不僅以單體的形式發(fā)揮作用，而且還可以二聚體或多聚體形式發(fā)揮不同于單體的作用。有報(bào)道指出， 型阿片受體可以與μ型阿片受體可以形成異源二聚體，并且促進(jìn)嗎啡誘導(dǎo)的μ型阿片受體的內(nèi)吞。 鑒于阿片受體是否內(nèi)吞和再循環(huán)是嗎啡誘導(dǎo)耐受的原因之一，那么有效地促進(jìn)阿片受體內(nèi)吞和再循環(huán)就可能抑制或?qū)箚岱饶褪艿男纬伞?我們以往的工作發(fā)現(xiàn)，慢性嗎啡處理可以抑制大鼠視上核催產(chǎn)素的合成和釋放，而特異性促進(jìn)催產(chǎn)素合成和釋放的鋰鹽可顯著抑制慢性嗎啡處理所導(dǎo)致的痛覺耐受，同時(shí)，這種效應(yīng)可以被催產(chǎn)素受體的拮抗劑所阻斷，提示催產(chǎn)素受體（OTR）可能參與了嗎啡鎮(zhèn)痛耐受的形成，但其作用機(jī)制目前尚不清楚。催產(chǎn)素受體與阿片受體同屬于G蛋白偶聯(lián)受體的A家族成員，但其偶聯(lián)的G蛋白不同。我們以往的研究發(fā)現(xiàn)，催產(chǎn)素受體可以以還原劑敏感的多聚體形式存在，其是否可與阿片受體形成異源二聚體參與阿片受體藥理學(xué)作用的調(diào)節(jié)目前尚未見報(bào)道。 本研究在我們以往的工作基礎(chǔ)上，觀察阿片受體和催產(chǎn)素受體異源二聚化對(duì)阿片受體內(nèi)吞的影響，探討G蛋白偶聯(lián)受體異源二聚化在嗎啡誘導(dǎo)的痛覺耐受中的作用，為開發(fā)和設(shè)計(jì)新的鎮(zhèn)痛藥物提供理論和實(shí)驗(yàn)依據(jù)。 以大鼠鞘內(nèi)給藥的方式建立嗎啡耐受模型，觀察催產(chǎn)素對(duì)慢性嗎啡處理所誘導(dǎo)耐受形成的影響，結(jié)果顯示嗎啡連續(xù)處理至第7天，大鼠形成對(duì)嗎啡的耐受，而催產(chǎn)素可延緩和抑制耐受的形成。免疫組化顯示，大鼠脊髓背角存在著MOR與OTR的共定位；免疫共沉淀顯示，脊髓中MOR與OTR存在相互作用。為了進(jìn)一步確認(rèn)MOR和OTR的相互作用和生理學(xué)意義，我們構(gòu)建了MOR-OTR共表達(dá)轉(zhuǎn)染細(xì)胞株，利用免疫共沉淀技術(shù)和雙分子熒光互補(bǔ)（BiFC）技術(shù)，確認(rèn)了兩者間存在的相互作用，同時(shí)采用SNAP/CLIP染色標(biāo)記的細(xì)胞影像技術(shù)，觀察催產(chǎn)素對(duì)于嗎啡誘導(dǎo)的MOR內(nèi)吞的影響。結(jié)果表明，低劑量的催產(chǎn)素與嗎啡聯(lián)合作用時(shí)，可以有效地促進(jìn)MOR的內(nèi)吞，而嗎啡和催產(chǎn)素單獨(dú)處理對(duì)MOR的內(nèi)吞沒有影響。為了研究MOR和OTR相互作用可能的功能區(qū)域，我們表達(dá)和純化了MOR的四個(gè)功能區(qū)段的GST融合蛋白，利用GST-Pull Down研究介導(dǎo)MOR與OTR相互作用的區(qū)段。結(jié)果顯示，μ型阿片受體第三個(gè)胞內(nèi)環(huán)可能參與了其與催產(chǎn)素受體之間的相互作用。 本實(shí)驗(yàn)所得結(jié)論如下： 1、鞘內(nèi)注射催產(chǎn)素，可以顯著提高嗎啡的鎮(zhèn)痛效率，延遲耐受的出現(xiàn)。 2、在大鼠脊髓存在著μ型阿片受體與催產(chǎn)素受體的相互作用。并且在脊髓背角，首次觀察到了μ型阿片受體與催產(chǎn)素受體的共定位。 3、在體外共表達(dá)體系中，μ型阿片受體與催產(chǎn)素受體有相互作用關(guān)系；低劑量的催產(chǎn)素可以有效促進(jìn)嗎啡誘導(dǎo)的μ型阿片受體的內(nèi)吞。 4、 μ型阿片受體的第三個(gè)胞內(nèi)環(huán)，參與了催產(chǎn)素受體與其的相互作用。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.61

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