TRPC通道在吗啡耐受和痛觉敏化中的作用研究

发布时间：2018-07-10 14:00

本文选题：吗啡 + 镇痛耐受　；参考：《昆明理工大学》2017年硕士论文

【摘要】：研究目的:吗啡是一种广泛应用于临床镇痛的阿片类受体激动剂。但长期反复地使用吗啡会导致患者出现耐受现象,并常伴有痛觉过敏和触诱发痛等并发症,极大的限制了吗啡的的临床应用。关于吗啡诱导的镇痛耐受和痛觉敏化的研究目前主要集中在中枢神经可塑性改变和阿片受体的脱敏与复敏受损两方面,但是对于吗啡引起的耐受及痛觉敏化的分子机理至今尚未完全清楚。本研究通过建立大鼠吗啡镇痛耐受和痛觉敏化模型,检测TRPC通道在脊髓的表达变化,并阻断TRPC通道表达,探究其在吗啡镇痛耐受和痛觉敏化中的作用机制。研究方法:(1)雄性SD大鼠随机分为对照组和吗啡组,每组6只。吗啡组大鼠连续7 d腹腔注射盐酸吗啡(10 mg/kg,2次/天);对照组腹腔注射等量生理盐水。分别于第1d、3d、5d、7d,在注射吗啡或生理盐水前后测定大鼠的机械痛阈和热痛阈。最后一次注射后16h,测试各组大鼠的机械痛阈值和热痛阈值,鞘内注射吗啡(5μg)30min后,重复上述行为学检测。随后截取大鼠腰段脊髓,采用qRT-PCR和Western blot检测对照组和吗啡组大鼠脊髓内TRPC通道各亚型mRNA和蛋白的表达变化。(2)雄性SD成年大鼠48只随机分为8组:对照组(Con组)、吗啡组(Mor 组)、2-APB 空白组、2-APB Ⅰ 组、2-APB Ⅱ 组、SKF 空白组、SKF Ⅰ 组、SKF Ⅱ组,每组6只。各组大鼠行鞘内置管手术,Mor组、2-APBI组、2-APBⅡ组、SKF Ⅰ组和SKF Ⅱ组大鼠连续7 d腹腔注射盐酸吗啡(10 mg/kg,2次/天);Con组、2-APB空白组和SKF空白组大鼠腹腔注射等量生理盐水。在此期间,经鞘内导管向2-APB Ⅰ组和2-APB Ⅱ组大鼠分别注射2-APB 0.1 μg和0.5 μg(1次/天),连续7d;向SKF Ⅰ组和SKF Ⅱ组大鼠分别注射SKF96365 0.1μg和0.5μg,(1次/天),连续7d;向2-APB空白组和SKF空白组大鼠分别注射0.5 μg 2-APB和SKF96365(1次/天),连续7d;Mor组和Con组大鼠经鞘内注射人工脑脊液20μg(1次/天),连续7d。最后一次盐水吗啡或生理盐水注射16h后,测试各组大鼠的机械痛阈值(g)和热痛阚值(s)。通过鞘内注射吗啡(5μg)30 min后,重复行为学检测。(3)雄性SD大鼠48只随机分为6组:S+V组、M+V组、S+Si组、S+Sc组、M+Si组、M+Sc组,每组6只。M+V组、M+Si组、M+Sc组大鼠腹腔注射盐酸吗啡(10mg/kg,2次/天),连续7d;S+V组、S+Si组、和S+Sc空白组大鼠腹腔注射等量生理盐水。在此期间,经鞘内向S+Si组和M+Si组大鼠注射TRPC6siRNA(20μl,1次/天),连续4d山向S+Sc组和M+Sc组大鼠鞘内注射scramblesiRNA(20μl,1次/天),连续4d;向S+V组和M+V组大鼠鞘内注射人工脑脊液(20μl,1次/天),连续4 d。最后一次盐酸吗啡或生理盐水注射16h后,测试各组大鼠的机械痛阈值(g)和热痛阈值(s)。通过鞘内留置导管注射吗啡(5μg)30min后,重复行为学检测。采用qRT-PCR、ELISA、Westemblot和免疫荧光技术检测各组大鼠脊髓内p-mTOR,PKCγ,nNOS、CaMKIIα、NF-κB、IL-1β,IL-6、TNF-α 以及 GFAP 和 Iba1 的表达变化。研究结果:(1)连续7 d腹腔注射吗啡能够诱导大鼠出现明显的慢性吗啡镇痛耐受。慢性吗啡处理能诱导大鼠脊髓TRPC1、TRPC3和TRPC6的mRNA和蛋白质水平表达增加;(2)鞘内给予TRPC通道阻断剂2-APB或SKF96365,可以抑制吗啡镇痛耐受和痛觉敏化的形成;(3)鞘内给予TRPC6siRNA能抑制吗啡镇痛耐受和痛觉敏化的形成,并抑制吗啡诱导的nNOS和CaMKIIα在脊髓的表达上调;脊髓TRPC6敲除抑制吗啡诱导的脊髓神经免疫激活。研究结论:TRPC6通道可能通过促进脊髓炎症反应和神经免疫反应参与吗啡诱导的镇痛耐受和痛觉敏化的形成。
[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.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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