P2X7受体介导大鼠慢性炎症性疼痛的脊髓机制的研究
发布时间：2018-11-09 09:16
【摘要】：研究目的 本研究拟采用药理学手段，，分子生物学和行为学手段，采用慢性炎症性大鼠，对P2X7受体传递和转化疼痛信号的作用及其机制进行分析，以便能找到更好的预防治疗慢性疼痛的理论依据。 研究方法 1．健康，活动正常，没有疾患的雄性Wistar成年大鼠按照对照随机原则分为如下三组：空白组（Naive组），假手术组（Sham组）和完全弗氏佐剂（CFA组），测定各组大鼠机械痛阈和热痛阈基础值后，选择左侧后肢，暴露大鼠踝关节，单关节炎（monoarthritis,MA）模型组抽取50μl完全弗氏佐剂注入关节腔内。假手术组用信的注射器抽取50μl生理盐水注入关节腔里。以上三组大鼠的机械痛阈和热痛阈需要在术后5天再次测定和记录。同时，同样数量的另外的三组模型动物在深度麻醉下解剖处死，取出腰膨大段脊髓背角组织，采用免疫印迹（Western blot）方法检测脊髓背角瞬时感受器电位香草酸受体l(transientreceptor potential vanilloid1, TRPV1)，P2X7R，配体门控型非选择性离子通道6(Purinergicrecepter P2X, ligand-gated ion channel6, P2X6R)，pCREB等的变化，并用免疫共沉淀（Co-immunoprecipitation，Co-Ip）检测P2X7R和PSD-95的相互作用。 2．雄性Wistar成年大鼠CFA后的6个不一样的时间点12h，24h，2d，3d，5d，7d，检测pERK，ERK在脊髓背角的表达数量高低情况，并以Sham组为基础，观察在CFA致炎后pERK，ERK在脊髓背角的变化趋势。 研究结果 1．术前三组大鼠机械痛阈和热痛阈基础值无显著性差异（P0.05），术后第5天，CFA组出现热缩足反射潜伏期和机械缩足反射阈值的降低，与假手术组或者与对侧比较，差异有显著性（P 0.01或0.05）。脊髓腰膨大处pCREB表达水平测定显示，术后5天MA组CREB的磷酸化增加了，与Sham组比较，差异有显著性（P 0.05）。而TRPV1，P2X7R,P2X6R的表达未见显著性变化（P0.05）；免疫共沉淀发现，CFA大鼠脊髓背角P2X7R和PSD-95的相互作用显著性增加。 2．我们在CFA后的不同时间点（12h,24h,2d,3d,5d,7d）,检测脊髓背角pERK,ERK的变化。结果发现，在CFA致炎后的12h，脊髓背角ERK的磷酸化即开始增加，并在随后的7d内，维持在较高的水平。 研究结论 1．脊髓背角P2X7R参与了大鼠CFA引起的慢性炎症性疼痛，有可能是P2X7R通过别的途径介导CREB，ERK的磷酸化来调节疼痛。 2.脊髓背角P2X7R参与了慢性炎症性疼痛发生发展的过程，更有可能是脊髓背角P2X7R通过和PSD-95的相互作用介导了CFA大鼠慢性炎症性疼痛的发生发展。
[Abstract]:Objective to investigate the effects and mechanisms of P2X7 receptors on the transmission and transformation of pain signals in chronic inflammatory rats by means of pharmacology, molecular biology and behavior. In order to find a better theoretical basis for the prevention and treatment of chronic pain. Adult male Wistar rats were randomly divided into three groups: blank group (Naive group), sham operation group (Sham group) and complete Freund's adjuvant group (CFA group). 2. After measuring the basic values of mechanical pain threshold and thermal pain threshold in each group, the ankle joint was exposed to the left hindlimb, and 50 渭 l complete Freund's adjuvant was injected into the articular cavity in the monoarthritis,MA model group. In the sham operation group, 50 渭 l of saline was injected into the articular cavity with a syringe. The mechanical and thermal pain thresholds of the above three groups need to be measured and recorded 5 days after operation. At the same time, the same number of other three groups of model animals were dissected and killed under deep anesthesia, the lumbar distended spinal dorsal horn tissue was removed, and the transient receptor l (transientreceptor potential vanilloid1, of vanillic acid receptor was detected by Western blot (Western blot) method. TRPV1, P2X7R, ligand gated nonselective ion channel 6 (Purinergicrecepter P2X, ligand-gated ion channel6, P2X6R), pCREB, etc.). The interaction between P2X7R and PSD-95 was detected by immunoprecipitation (Co-immunoprecipitation,Co-Ip). 2.The expression of pERK,ERK in spinal dorsal horn was detected at 6 different time points after CFA of male Wistar adult rats at 12 h, 24 h, 2 d, 3 d and 5 d for 7 days. Based on Sham group, pERK, was observed after CFA inflammation. The change trend of ERK in the dorsal horn of spinal cord. Results 1. There was no significant difference in mechanical pain threshold and thermal pain threshold between the three groups before operation (P0.05). On the 5th day after operation, the latent period of thermal contraction foot reflex and the decrease of mechanical foot reflex threshold appeared in CFA group. There was significant difference between sham operation group and contralateral side (P 0.01 or 0.05). The expression of pCREB in spinal cord lumbar expansion showed that the phosphorylation of CREB was increased in MA group 5 days after operation, which was significantly different from that in Sham group (P 0.05). The expression of TRPV1,P2X7R,P2X6R did not change significantly (P0.05), and the interaction between P2X7R and PSD-95 in the spinal dorsal horn of CFA rats was significantly increased by co-immunoprecipitation. 2. The changes of pERK,ERK in the dorsal horn of spinal cord were detected at different time points after CFA (12 h, 24 h, 2 d, 3 d, 5 d, 7 d). The results showed that the phosphorylation of ERK in the dorsal horn of spinal cord began to increase 12 hours after CFA and remained at a higher level within the following 7 days. Spinal dorsal horn P2X7R is involved in chronic inflammatory pain induced by CFA in rats. It may be that P2X7R mediates the phosphorylation of CREB,ERK in other ways to regulate pain. 2. 2. P2X7R in the dorsal horn of spinal cord is involved in the development of chronic inflammatory pain, and it is more likely that P2X7R mediates the development of chronic inflammatory pain in CFA rats through the interaction with PSD-95.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R402
本文编号：2319996
[Abstract]:Objective to investigate the effects and mechanisms of P2X7 receptors on the transmission and transformation of pain signals in chronic inflammatory rats by means of pharmacology, molecular biology and behavior. In order to find a better theoretical basis for the prevention and treatment of chronic pain. Adult male Wistar rats were randomly divided into three groups: blank group (Naive group), sham operation group (Sham group) and complete Freund's adjuvant group (CFA group). 2. After measuring the basic values of mechanical pain threshold and thermal pain threshold in each group, the ankle joint was exposed to the left hindlimb, and 50 渭 l complete Freund's adjuvant was injected into the articular cavity in the monoarthritis,MA model group. In the sham operation group, 50 渭 l of saline was injected into the articular cavity with a syringe. The mechanical and thermal pain thresholds of the above three groups need to be measured and recorded 5 days after operation. At the same time, the same number of other three groups of model animals were dissected and killed under deep anesthesia, the lumbar distended spinal dorsal horn tissue was removed, and the transient receptor l (transientreceptor potential vanilloid1, of vanillic acid receptor was detected by Western blot (Western blot) method. TRPV1, P2X7R, ligand gated nonselective ion channel 6 (Purinergicrecepter P2X, ligand-gated ion channel6, P2X6R), pCREB, etc.). The interaction between P2X7R and PSD-95 was detected by immunoprecipitation (Co-immunoprecipitation,Co-Ip). 2.The expression of pERK,ERK in spinal dorsal horn was detected at 6 different time points after CFA of male Wistar adult rats at 12 h, 24 h, 2 d, 3 d and 5 d for 7 days. Based on Sham group, pERK, was observed after CFA inflammation. The change trend of ERK in the dorsal horn of spinal cord. Results 1. There was no significant difference in mechanical pain threshold and thermal pain threshold between the three groups before operation (P0.05). On the 5th day after operation, the latent period of thermal contraction foot reflex and the decrease of mechanical foot reflex threshold appeared in CFA group. There was significant difference between sham operation group and contralateral side (P 0.01 or 0.05). The expression of pCREB in spinal cord lumbar expansion showed that the phosphorylation of CREB was increased in MA group 5 days after operation, which was significantly different from that in Sham group (P 0.05). The expression of TRPV1,P2X7R,P2X6R did not change significantly (P0.05), and the interaction between P2X7R and PSD-95 in the spinal dorsal horn of CFA rats was significantly increased by co-immunoprecipitation. 2. The changes of pERK,ERK in the dorsal horn of spinal cord were detected at different time points after CFA (12 h, 24 h, 2 d, 3 d, 5 d, 7 d). The results showed that the phosphorylation of ERK in the dorsal horn of spinal cord began to increase 12 hours after CFA and remained at a higher level within the following 7 days. Spinal dorsal horn P2X7R is involved in chronic inflammatory pain induced by CFA in rats. It may be that P2X7R mediates the phosphorylation of CREB,ERK in other ways to regulate pain. 2. 2. P2X7R in the dorsal horn of spinal cord is involved in the development of chronic inflammatory pain, and it is more likely that P2X7R mediates the development of chronic inflammatory pain in CFA rats through the interaction with PSD-95.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R402
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1 李娜娜;P2X7受体介导大鼠慢性炎症性疼痛的脊髓机制的研究[D];山西医科大学;2014年
本文编号：2319996
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