本文選題：嗎啡 + 鎮(zhèn)痛耐受��； 參考：《昆明理工大學(xué)》2017年碩士論文

【摘要】：研究目的:嗎啡是一種廣泛應(yīng)用于臨床鎮(zhèn)痛的阿片類受體激動(dòng)劑。但長期反復(fù)地使用嗎啡會(huì)導(dǎo)致患者出現(xiàn)耐受現(xiàn)象,并常伴有痛覺過敏和觸誘發(fā)痛等并發(fā)癥,極大的限制了嗎啡的的臨床應(yīng)用。關(guān)于嗎啡誘導(dǎo)的鎮(zhèn)痛耐受和痛覺敏化的研究目前主要集中在中樞神經(jīng)可塑性改變和阿片受體的脫敏與復(fù)敏受損兩方面,但是對(duì)于嗎啡引起的耐受及痛覺敏化的分子機(jī)理至今尚未完全清楚。本研究通過建立大鼠嗎啡鎮(zhèn)痛耐受和痛覺敏化模型,檢測(cè)TRPC通道在脊髓的表達(dá)變化,并阻斷TRPC通道表達(dá),探究其在嗎啡鎮(zhèn)痛耐受和痛覺敏化中的作用機(jī)制。研究方法:(1)雄性SD大鼠隨機(jī)分為對(duì)照組和嗎啡組,每組6只。嗎啡組大鼠連續(xù)7 d腹腔注射鹽酸嗎啡(10 mg/kg,2次/天);對(duì)照組腹腔注射等量生理鹽水。分別于第1d、3d、5d、7d,在注射嗎啡或生理鹽水前后測(cè)定大鼠的機(jī)械痛閾和熱痛閾。最后一次注射后16h,測(cè)試各組大鼠的機(jī)械痛閾值和熱痛閾值,鞘內(nèi)注射嗎啡(5μg)30min后,重復(fù)上述行為學(xué)檢測(cè)。隨后截取大鼠腰段脊髓,采用qRT-PCR和Western blot檢測(cè)對(duì)照組和嗎啡組大鼠脊髓內(nèi)TRPC通道各亞型mRNA和蛋白的表達(dá)變化。(2)雄性SD成年大鼠48只隨機(jī)分為8組:對(duì)照組(Con組)、嗎啡組(Mor 組)、2-APB 空白組、2-APB Ⅰ 組、2-APB Ⅱ 組、SKF 空白組、SKF Ⅰ 組、SKF Ⅱ組,每組6只。各組大鼠行鞘內(nèi)置管手術(shù),Mor組、2-APBI組、2-APBⅡ組、SKF Ⅰ組和SKF Ⅱ組大鼠連續(xù)7 d腹腔注射鹽酸嗎啡(10 mg/kg,2次/天);Con組、2-APB空白組和SKF空白組大鼠腹腔注射等量生理鹽水。在此期間,經(jīng)鞘內(nèi)導(dǎo)管向2-APB Ⅰ組和2-APB Ⅱ組大鼠分別注射2-APB 0.1 μg和0.5 μg(1次/天),連續(xù)7d;向SKF Ⅰ組和SKF Ⅱ組大鼠分別注射SKF96365 0.1μg和0.5μg,(1次/天),連續(xù)7d;向2-APB空白組和SKF空白組大鼠分別注射0.5 μg 2-APB和SKF96365(1次/天),連續(xù)7d;Mor組和Con組大鼠經(jīng)鞘內(nèi)注射人工腦脊液20μg(1次/天),連續(xù)7d。最后一次鹽水嗎啡或生理鹽水注射16h后,測(cè)試各組大鼠的機(jī)械痛閾值(g)和熱痛闞值(s)。通過鞘內(nèi)注射嗎啡(5μg)30 min后,重復(fù)行為學(xué)檢測(cè)。(3)雄性SD大鼠48只隨機(jī)分為6組:S+V組、M+V組、S+Si組、S+Sc組、M+Si組、M+Sc組,每組6只。M+V組、M+Si組、M+Sc組大鼠腹腔注射鹽酸嗎啡(10mg/kg,2次/天),連續(xù)7d;S+V組、S+Si組、和S+Sc空白組大鼠腹腔注射等量生理鹽水。在此期間,經(jīng)鞘內(nèi)向S+Si組和M+Si組大鼠注射TRPC6siRNA(20μl,1次/天),連續(xù)4d山向S+Sc組和M+Sc組大鼠鞘內(nèi)注射scramblesiRNA(20μl,1次/天),連續(xù)4d;向S+V組和M+V組大鼠鞘內(nèi)注射人工腦脊液(20μl,1次/天),連續(xù)4 d。最后一次鹽酸嗎啡或生理鹽水注射16h后,測(cè)試各組大鼠的機(jī)械痛閾值(g)和熱痛閾值(s)。通過鞘內(nèi)留置導(dǎo)管注射嗎啡(5μg)30min后,重復(fù)行為學(xué)檢測(cè)。采用qRT-PCR、ELISA、Westemblot和免疫熒光技術(shù)檢測(cè)各組大鼠脊髓內(nèi)p-mTOR,PKCγ,nNOS、CaMKIIα、NF-κB、IL-1β,IL-6、TNF-α 以及 GFAP 和 Iba1 的表達(dá)變化。研究結(jié)果:(1)連續(xù)7 d腹腔注射嗎啡能夠誘導(dǎo)大鼠出現(xiàn)明顯的慢性嗎啡鎮(zhèn)痛耐受。慢性嗎啡處理能誘導(dǎo)大鼠脊髓TRPC1、TRPC3和TRPC6的mRNA和蛋白質(zhì)水平表達(dá)增加;(2)鞘內(nèi)給予TRPC通道阻斷劑2-APB或SKF96365,可以抑制嗎啡鎮(zhèn)痛耐受和痛覺敏化的形成;(3)鞘內(nèi)給予TRPC6siRNA能抑制嗎啡鎮(zhèn)痛耐受和痛覺敏化的形成,并抑制嗎啡誘導(dǎo)的nNOS和CaMKIIα在脊髓的表達(dá)上調(diào);脊髓TRPC6敲除抑制嗎啡誘導(dǎo)的脊髓神經(jīng)免疫激活。研究結(jié)論:TRPC6通道可能通過促進(jìn)脊髓炎癥反應(yīng)和神經(jīng)免疫反應(yīng)參與嗎啡誘導(dǎo)的鎮(zhèn)痛耐受和痛覺敏化的形成。
[Abstract]:Objective: morphine is a kind of opioid agonist widely used in clinical analgesia. However, the long-term use of morphine can lead to the occurrence of tolerance, often accompanied by complications such as hyperalgesia and tactile pain, which greatly restrict the clinical application of morphine. The study of morphine induced pain tolerance and pain sensitization At present, two aspects of central nerve plasticity and opioid receptor desensitization and hypersensitivity are mainly concentrated, but the molecular mechanism of morphine induced tolerance and sensitization is not completely clear. In this study, the changes in the expression of TRPC channel in the spinal cord were detected by the establishment of morphine tolerance and pain sensitization model in rats. The expression of TRPC channel was used to explore the mechanism of its effect on morphine analgesia and pain sensitization. (1) the male SD rats were randomly divided into control group and morphine group, 6 rats in each group. The morphine group was injected with morphine (10 mg/kg, 2 times) intraperitoneally for 7 d in morphine group, and the control group was injected with equal amount of saline in the abdominal cavity, 1D, 3D, 5D, 7d, respectively. The mechanical pain threshold and thermal pain threshold of rats were measured before and after morphine or physiological saline. After the last injection, 16h was used to test the mechanical pain threshold and the threshold of heat pain in each group. After intrathecal injection of morphine (5 u g) 30min, the above behavioral test was repeated. Then the spinal cord of the rats was intercepted, and qRT-PCR and Western blot were used to detect the control group and the morphine group. (2) 48 male SD adult rats were randomly divided into 8 groups: control group (group Con), morphine group (group Mor), 2-APB blank group, 2-APB I group, 2-APB II group, SKF blank group, SKF I group, SKF II group, 6 rats in each group. The rats in group F I and group SKF II were intraperitoneally injected with morphine (10 mg/kg, 2 times) for 7 d; Con group, 2-APB blank group and SKF blank group were injected with equal amount of saline. During this period, the intrabasal catheter to 2-APB I and 2-APB II rats were injected with 2-APB 0.1 Mu G and 0.5 micron g (1 times / day). Rats were injected with SKF96365 0.1 mu g and 0.5 u g, (1 times / day) and continuous 7d. 0.5 mu g 2-APB and SKF96365 (1 times / day) were injected into 2-APB blank group and SKF blank group, and 20 mu of artificial cerebrospinal fluid (1 / day) was injected into the sheath of Mor and Con group (1 times / day). After the last saline injection of morphine or saline, each group was tested. The mechanical pain threshold (g) and the thermal threshold value (s) of the rats. After the intrathecal injection of morphine (5 u g) 30 min, the repetitive behavior test was detected. (3) 48 male SD rats were randomly divided into 6 groups: S+V group, M+V group, S+Si group, S+Sc group, M+Si group, M+Sc group, each group of 6 rats were injected with morphine hydrochloride (2 times / day), consecutive Rats in the S+Sc blank group were intraperitoneally injected with equal amount of saline. During this period, TRPC6siRNA (20 mu L, 1 times) were injected into group S+Si and M+Si in the sheath of the sheath, and scramblesiRNA (20 mu L, 1 times / day) in the S+Sc group and M+Sc group were injected into the S+Sc group and M+Sc group. After the final injection of morphine or saline 16h, the mechanical pain threshold (g) and the heat pain threshold (s) were tested in each group. After injection of morphine (5 mu g) 30min in the sheath, the repeated behavioral tests were detected. QRT-PCR, ELISA, Westemblot and immunofluorescence were used to detect p-mTOR, PKC, nNOS, CaMKII alpha and CaMKII alpha in the spinal cord of rats. The changes in expression of 1 beta, IL-6, TNF- A and GFAP and Iba1. Results: (1) a continuous 7 d intraperitoneal injection of morphine can induce obvious chronic morphine tolerance in rats. Chronic morphine treatment can induce TRPC1, mRNA and protein levels of TRPC1, TRPC3 and TRPC6 in rat spinal cord, and (2) TRPC channel blockers in the sheath, 2-APB or SKF9636. 5, it can inhibit the formation of morphine pain tolerance and pain sensitization; (3) TRPC6siRNA in the sheath can inhibit the formation of morphine analgesia and pain sensitization, and inhibit the up-regulated expression of morphine induced nNOS and CaMKII alpha in the spinal cord; TRPC6 knockout in the spinal cord inhibits morphine induced neuroimmunological activation of the spinal cord. The conclusion: the TRPC6 channel may pass through the study. Spinal cord inflammatory response and neuroimmune response are involved in morphine induced analgesia tolerance and pain sensitization.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R965

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本文編號(hào)：2113593