本文選題：嗎啡耐受 + 痛覺過敏��； 參考：《鄭州大學》2016年碩士論文

【摘要】：實驗背景與目的嗎啡是阿片類受體激動劑,是應(yīng)用于臨床鎮(zhèn)痛的金標準,但是在多次反復使用后,會產(chǎn)生鎮(zhèn)痛耐受以及停藥后的痛覺過敏現(xiàn)象。對嗎啡耐受雖然已經(jīng)進行了大量研究,但其產(chǎn)生機制迄今仍不十分清楚。最近研究發(fā)現(xiàn),反復大量使用嗎啡后,在神經(jīng)系統(tǒng)會引起一些適應(yīng)性變化,包括阿片類受體的脫敏,內(nèi)化,下調(diào)以及磷酸化現(xiàn)象或者與其他受體異源二聚化。慢性阿片類物質(zhì)的接觸會引起神經(jīng)膠質(zhì)細胞以及谷氨酸受體的活化和促炎細胞因子的釋放等。其中細胞因子的釋放可能是引起嗎啡鎮(zhèn)痛耐受和痛覺過敏產(chǎn)生的主要原因。高遷移率族蛋白1(High mobility group box protein 1,HMGB1)是一種類似于細胞因子的炎性介質(zhì),屬于HMGB家族。HMGB1廣泛存在于哺乳動物的細胞中,在接受刺激時可以從免疫和非免疫細胞當中釋放到細胞外環(huán)境,從而引起各種炎癥反應(yīng)。近年來的研究發(fā)現(xiàn),周圍神經(jīng)損傷可引起HMGB1在背根神經(jīng)節(jié)(Dorsal Root Ganglia,DRG)和脊髓背角的表達變化,阻斷HMGB1的作用可減輕大鼠神經(jīng)病理性疼痛。目前,多數(shù)國內(nèi)外學者認為神經(jīng)病理性痛和嗎啡耐受有部分相似的發(fā)生機制。雖然HMGB1在病理性疼痛中的作用已經(jīng)取得了明確的實驗結(jié)果,但HMGB1在嗎啡耐受中的作用和具體的分子機制迄今未見報道。本研究的主要目的就是通過動物實驗證明HMGB1在嗎啡耐受形成過程的作用。為此本實驗首先采用多次(每天2次,共6天)鞘內(nèi)微量注射嗎啡(10μg/10μl)建立大鼠嗎啡鎮(zhèn)痛耐受模型,通過檢測大鼠甩尾潛伏期(Tail-flick la tency)觀測嗎啡鎮(zhèn)痛作用的變化,通過測試大鼠機械刺激撤足閾值(Paw With drawal Threshold,PWT)和熱刺激撤足潛伏期(Paw Withdrawal Latency,PWL)評估停用嗎啡后大鼠的痛行為學變化。實驗過程通過在嗎啡耐受形成的不同時段(嗎啡耐受形成前和嗎啡耐受形成后)鞘內(nèi)注射Glycyrrhizin(GL)(HMGB1的小分子抑制劑)或HMGB1 si RNA,結(jié)合以上行為學測試,以觀察阻斷HM GB1的作用或抑制HMGB1的合成對嗎啡耐受形成的影響。實驗還通過Western Blot法進一步觀察了在嗎啡耐受形成過程HMGB1在DRG和脊髓背角的表達變化以及鞘內(nèi)注射HMGB1 si RNA對HMGB1表達的影響;通過免疫熒光雙染色法觀察了嗎啡誘導的HMGB1在大鼠DRG和脊髓背角表達的細胞類型。實驗結(jié)果將對進一步闡明嗎啡耐受形成的機制提供理論支持,同時對臨床防治嗎啡耐受的產(chǎn)生有一定實際參考價值。實驗結(jié)果1.嗎啡耐受形成過程HMGB1在大鼠DRG和脊髓背角的表達變化以及表達的細胞類型大鼠鞘內(nèi)注射嗎啡10μg/10μl一天兩次,連續(xù)6天注射。在給藥的第5天嗎啡的鎮(zhèn)痛效率在20%左右(**P0.01),在第7天嗎啡的鎮(zhèn)痛效率幾乎為0(與注射生理鹽水組比較,***P0.001)。嗎啡停藥后第8天會引起大鼠的雙側(cè)機械痛覺超敏和熱痛覺過敏(與注射生理鹽水組比較,***P0.001)。West ern Blot檢測發(fā)現(xiàn)鞘內(nèi)注射嗎啡引起了HMGB1在脊髓背角和DRG中的表達量的明顯升高(與注射生理鹽水組比較,**P0.001),通過免疫熒光雙染發(fā)現(xiàn)H MGB1在脊髓背角中與神經(jīng)元、星形膠質(zhì)細胞以及小膠質(zhì)細胞均有共定位,在背根神經(jīng)節(jié)中HMGB1與A類C類神經(jīng)元以及衛(wèi)星樣膠質(zhì)細胞共定位。2.鞘內(nèi)預注射HMGB1小分子抑制劑Glycyrrhizin部分抑制了嗎啡耐受的形成鞘內(nèi)注射GL組和注射溶劑組(Vehicle,Veh)相比較,在嗎啡注射的第5天(***P0.001)和第7天(**P0.01,***P0.001)嗎啡的鎮(zhèn)痛效率有了明顯的提升。停藥后第8天大鼠的PWT和PWL和溶劑組相比較均有一定程度的提高(*P0.05,**P0.01)。結(jié)果提示鞘內(nèi)注射GL延緩了嗎啡耐受的形成,同時部分減輕了嗎啡停藥后所引起的痛覺過敏,作用具有一定的時間和劑量依賴性。3.嗎啡耐受模型建立后鞘內(nèi)預注射GL能夠部分逆轉(zhuǎn)嗎啡耐受癥狀大鼠嗎啡耐受模型建立后第7天,在鞘內(nèi)注射嗎啡的同時,鞘內(nèi)注射GL(提前30 min,50μg/10μl,每天一次,共5天)。結(jié)果發(fā)現(xiàn)在從第7天開始到第11天鞘內(nèi)注射Gl組和溶劑組相比較,鞘內(nèi)注射GL組的嗎啡鎮(zhèn)痛效率逐漸升高(**P0.01,***P0.001)。在第12天停止給藥后大鼠的PWT和PWL和溶劑組相比較均有明顯的升高(**P0.01,***P0.001)。結(jié)果表明鞘內(nèi)注射GL可部分逆轉(zhuǎn)已經(jīng)形成的嗎啡鎮(zhèn)痛耐受。4.鞘內(nèi)注射HMGB1 si RNA可以延緩嗎啡耐受的形成鞘內(nèi)注射嗎啡+HMGB1 si RNA組和嗎啡+生理鹽水組、嗎啡+HMGB1 Scra mble si RNA組以及嗎啡+Transfection Regant組相比較,在嗎啡注射的第5天(***P0.001)和第7天(**P0.01)嗎啡的鎮(zhèn)痛效率有了明顯的提升。停藥后第8天大鼠的PWT和PWl和溶劑組相比較均有一定程度的提高(*P0.05,**P0.01)。HMGB1 Scramble si RNA組和Transfection Regant組行為學結(jié)果和嗎啡+生理鹽水組相比較無統(tǒng)計學差異。Western Blot結(jié)果顯示,嗎啡+HMGB1 si RN A組與生理鹽水組、嗎啡+HMGB1 Scramble si RNA組以及嗎啡+Transfection R egant組相比較,脊髓背角HMGB1的表達量最低(**P0.01),表明鞘內(nèi)注射HMGB1 si RNA有效抑制了HMGB1在大鼠脊髓背角的合成。5.運動功能檢測發(fā)現(xiàn)鞘內(nèi)連續(xù)注射嗎啡、GL以及HMGB1 si RNA對大鼠的運動功能沒有影響大鼠完成機械撤足閾值和熱撤足潛伏期測試后,進行運動功能檢測,通過放置反射、抓握反射、翻正反射三種測試方式,與對照組比較,給藥組大鼠沒有產(chǎn)生運動功能障礙。實驗結(jié)論抑制大鼠脊髓HMGB1的表達或阻斷HMGB1的作用,可以有效延緩嗎啡引起的鎮(zhèn)痛耐受以及停用嗎啡后的機械刺激痛覺超敏和熱刺激痛覺過敏癥狀。
[Abstract]:Background and objective morphine is opioid receptor agonist, which is the gold standard for clinical analgesia, but it will produce analgesic tolerance and hyperalgesia after repeated use. Although many studies have been done on morphine tolerance, the production mechanism is still not very clear. After the use of morphine, some adaptive changes are caused in the nervous system, including desensitization, internalization, downregulation and phosphorylation of opioid receptors, or heterogenous fusion with other receptors. The contact of chronic opioids may cause the activation of neuroglial cells and the activation of glutamate receptors and the release of proinflammatory cytokines. The release of cytokines may be the main cause of morphine tolerance and hyperalgesia. High mobility group protein 1 (High mobility group box protein 1, HMGB1) is an inflammatory medium similar to cytokine, which belongs to the HMGB family.HMGB1 widely existed in mammalian cells. Non immune cells are released into the extracellular environment and cause various inflammatory responses. Recent studies have found that peripheral nerve damage can cause changes in the expression of HMGB1 in the dorsal root ganglion (Dorsal Root Ganglia, DRG) and the dorsal horn of the spinal cord. Blocking the action of HMGB1 can reduce the neuropathic pain in rats. Most domestic and foreign scholars believe that the neuropathic pain can be reduced. Neuropathic pain and morphine tolerance are partly similar to the mechanism of morphine tolerance. Although the role of HMGB1 in pathological pain has been made clear, the role of HMGB1 in morphine tolerance and specific molecular mechanisms have not been reported. The main purpose of this study was to demonstrate that HMGB1 was in morphine tolerance through animal experiments. In this experiment, a rat model of morphine analgesic tolerance was established by microinjection of morphine (10 mu g/10 Mu L) in the sheath for 6 days (2 times a day for a total of 6 days). The changes in the analgesic effect of morphine were measured by the test of rat tail flick latency (Tail-flick La tency), and the threshold of the withdrawal of morphine (Paw With drawal Thresh) was tested. Old, PWT) and thermal stimulation of the foot latency (Paw Withdrawal Latency, PWL) assessment of the changes in the pain behavior of rats after the discontinuation of morphine. The experimental process was combined with the intrathecal injection of Glycyrrhizin (GL) (HMGB1's small molecule inhibitor) or HMGB1 Si RNA, in the different periods of morphine tolerance formation (morphine tolerance formation before and after morphine tolerance formation). The effects of blocking HM GB1 or inhibiting the synthesis of HMGB1 on the formation of morphine tolerance were observed. The effects of HMGB1 on the expression of HMGB1 in DRG and spinal dorsal horn in morphine tolerance formation and the effect of HMGB1 Si RNA on HMGB1 expression in the sheath were further observed by Western Blot; immunofluorescence double staining was used. The expression of morphine induced HMGB1 in rat DRG and dorsal horn of spinal cord was observed. The results of the experiment will provide theoretical support for further elucidate the mechanism of morphine tolerance formation, and have some practical reference value for the production of morphine tolerance in clinical. 1. the experimental results of morphine tolerance formation in rat DRG and spinal cord dorsal The changes in the expression of the angle and the expression of the cell type in the rat sheath were injected with morphine 10 g/10 Mu l a day two times a day for 6 days. The analgesic efficiency of morphine was about 20% (**P0.01) on the fifth day of the administration, and the analgesic efficiency of morphine was almost 0 in seventh days (compared with that of the injected saline group, ***P0.001). The morphine was stopped at eighth days after the withdrawal of morphine. The lateral mechanical pain hypersensitivity and hyperalgesia (compared with the injected physiological saline group, ***P0.001).West ERN Blot detected that intrathecal morphine induced a significant increase in HMGB1 expression in the dorsal horn and DRG of the spinal cord (compared with the injected physiological saline group, **P0.001), and found that H MGB1 was in the dorsal horn of the spinal cord and neurons by immunofluorescence double staining. Astrocytes and microglia have co localization. In the dorsal root ganglia, HMGB1, a class a C neuron and the satellite like glial cells are Co located in the.2. sheath for the pre injection of the HMGB1 small molecule inhibitor Glycyrrhizin, which inhibits the formation of morphine tolerance in the intrathecal GL group and the injection solvent group (Vehicle, Veh), in morphine injection. The analgesic efficiency of morphine, fifth days (***P0.001) and seventh days (**P0.01, ***P0.001), was significantly improved. The PWT and PWL in the rats were improved to a certain extent on the eighth day after the withdrawal (*P0.05, **P0.01). The results suggested that intrathecal GL delayed the formation of morphine tolerance and partly alleviated morphine induced withdrawal. Hyperalgesia, the effect has a certain time and dose dependent.3. morphine tolerance model after the establishment of intrathecal preinjection of GL can partially reverse morphine tolerance model rats after the establishment of morphine tolerance model seventh days after intrathecal injection of morphine, intrathecal injection of GL (early 30 min, 50 mu g/10 L, a total of 5 days a day). Results found in from seventh The analgesic efficiency of morphine in the intrathecal GL group was increased gradually (**P0.01, ***P0.001) in the intrathecal injection of Gl and the solvent group at the beginning of the day to eleventh days. The PWT and PWL and the PWL in the rats were significantly increased (**P0.01, ***P0.001) after the twelfth day of withdrawal (**P0.01, ***P0.001). The results showed that intrathecal GL partly reversed the morphine that had been formed. The intrathecal injection of HMGB1 Si RNA for analgesic tolerance to.4. could delay morphine tolerance in the formation of morphine +HMGB1 Si RNA group and morphine + physiological saline group. Compared with morphine +HMGB1 Scra mble Si RNA group and morphine group, the analgesic efficiency of morphine fifth days and seventh days of morphine was obvious. On the eighth day after stopping the drug, the PWT and PWl and the solvent group had a certain degree of improvement (*P0.05, **P0.01).HMGB1 Scramble Si RNA group and Transfection Regant group, and there was no statistical difference between the morphine + physiological saline group and the morphine + physiological saline group. Compared with the +HMGB1 Scramble Si RNA group and the morphine +Transfection R egant group, the expression of HMGB1 in the dorsal horn of the spinal cord was the lowest (**P0.01). It showed that the intrathecal HMGB1 Si RNA effectively inhibited the synthesis of the spinal dorsal horn of the rat. After the rats completed the test of the threshold of mechanical withdrawal and the incubation period of the heat withdrawal, the exercise function test was carried out, and three kinds of test methods were carried out by placing reflection, grasping reflex and turning positive reflex. Compared with the control group, the rats did not produce motor dysfunction. The experimental conclusion inhibited the expression of HMGB1 in the spinal cord of rats or blocked the effect of HMGB1. The analgesic effect of morphine is delayed, and the symptoms of hyperalgesia and heat induced hyperalgesia after morphine withdrawal are delayed.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R402

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