P2X7受體介導(dǎo)大鼠慢性炎癥性疼痛的脊髓機制的研究
發(fā)布時間：2018-11-09 09:16
【摘要】：研究目的 本研究擬采用藥理學(xué)手段，，分子生物學(xué)和行為學(xué)手段，采用慢性炎癥性大鼠，對P2X7受體傳遞和轉(zhuǎn)化疼痛信號的作用及其機制進行分析，以便能找到更好的預(yù)防治療慢性疼痛的理論依據(jù)。 研究方法 1．健康，活動正常，沒有疾患的雄性Wistar成年大鼠按照對照隨機原則分為如下三組：空白組（Naive組），假手術(shù)組（Sham組）和完全弗氏佐劑（CFA組），測定各組大鼠機械痛閾和熱痛閾基礎(chǔ)值后，選擇左側(cè)后肢，暴露大鼠踝關(guān)節(jié)，單關(guān)節(jié)炎（monoarthritis,MA）模型組抽取50μl完全弗氏佐劑注入關(guān)節(jié)腔內(nèi)。假手術(shù)組用信的注射器抽取50μl生理鹽水注入關(guān)節(jié)腔里。以上三組大鼠的機械痛閾和熱痛閾需要在術(shù)后5天再次測定和記錄。同時，同樣數(shù)量的另外的三組模型動物在深度麻醉下解剖處死，取出腰膨大段脊髓背角組織，采用免疫印跡（Western blot）方法檢測脊髓背角瞬時感受器電位香草酸受體l(transientreceptor potential vanilloid1, TRPV1)，P2X7R，配體門控型非選擇性離子通道6(Purinergicrecepter P2X, ligand-gated ion channel6, P2X6R)，pCREB等的變化，并用免疫共沉淀（Co-immunoprecipitation，Co-Ip）檢測P2X7R和PSD-95的相互作用。 2．雄性Wistar成年大鼠CFA后的6個不一樣的時間點12h，24h，2d，3d，5d，7d，檢測pERK，ERK在脊髓背角的表達數(shù)量高低情況，并以Sham組為基礎(chǔ)，觀察在CFA致炎后pERK，ERK在脊髓背角的變化趨勢。 研究結(jié)果 1．術(shù)前三組大鼠機械痛閾和熱痛閾基礎(chǔ)值無顯著性差異（P0.05），術(shù)后第5天，CFA組出現(xiàn)熱縮足反射潛伏期和機械縮足反射閾值的降低，與假手術(shù)組或者與對側(cè)比較，差異有顯著性（P 0.01或0.05）。脊髓腰膨大處pCREB表達水平測定顯示，術(shù)后5天MA組CREB的磷酸化增加了，與Sham組比較，差異有顯著性（P 0.05）。而TRPV1，P2X7R,P2X6R的表達未見顯著性變化（P0.05）；免疫共沉淀發(fā)現(xiàn)，CFA大鼠脊髓背角P2X7R和PSD-95的相互作用顯著性增加。 2．我們在CFA后的不同時間點（12h,24h,2d,3d,5d,7d）,檢測脊髓背角pERK,ERK的變化。結(jié)果發(fā)現(xiàn)，在CFA致炎后的12h，脊髓背角ERK的磷酸化即開始增加，并在隨后的7d內(nèi)，維持在較高的水平。 研究結(jié)論 1．脊髓背角P2X7R參與了大鼠CFA引起的慢性炎癥性疼痛，有可能是P2X7R通過別的途徑介導(dǎo)CREB，ERK的磷酸化來調(diào)節(jié)疼痛。 2.脊髓背角P2X7R參與了慢性炎癥性疼痛發(fā)生發(fā)展的過程，更有可能是脊髓背角P2X7R通過和PSD-95的相互作用介導(dǎo)了CFA大鼠慢性炎癥性疼痛的發(fā)生發(fā)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R402
本文編號：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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R402
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1 李娜娜;P2X7受體介導(dǎo)大鼠慢性炎癥性疼痛的脊髓機制的研究[D];山西醫(yī)科大學(xué);2014年
本文編號：2319996
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