小胶质细胞P2X4受体促进慢性内脏痛脊髓中枢的敏化

发布时间：2018-06-17 22:59

本文选题：慢性内脏痛 + 脊髓背角　；参考：《福建医科大学》2015年硕士论文

【摘要】：目的:探究小胶质细胞P2X4受体对慢性功能性内脏痛脊髓中枢敏化作用,为慢性内脏痛的药物治疗提供新的作用靶点和治疗方向。方法:新生SD大鼠出生第3~21天,每天固定时间给予3小时母婴分离,建立肠易激综合征(IBS)慢内脏痛大鼠模型。通过检测大鼠腹外斜肌对结直肠扩张的放电反应来评估IBS模型建立是否成功,并记录大鼠腹外斜肌在鞘内给予P2X4受体阻断剂5-BDBD、激动剂C5-TDS前后对于结直肠扩张的放电幅值;采用Westernblot方法检测大鼠脊髓P2X4受体以及脑神经营养因子(BDNF)的表达情况;借助免疫组化的方法检测大鼠脊髓背角离子钙接头蛋白1(Iba-1)表达情况来观察小胶质细胞的活化状态;通过免疫荧光双标方法观察大鼠脊髓背角P2X4受体与Iba-1蛋白表达部位是否一致来确认P2X4受体是否在小胶质细胞上表达。结果:与正常大鼠相比较,IBS大鼠腹外斜肌对结直肠扩张刺激反应显著增强(p0.05);鞘内给予P2X4受体阻断剂5-BDBD可抑制IBS和正常大鼠腹外斜肌对结直肠扩张的放电反应,同等剂量下,其对IBS大鼠的阻断反应更明显(p0.05),而且在IBS大鼠该阻断剂表现出明显的剂量依赖效应;鞘内给予P2X4激动剂C5-TDS后,IBS和正常大鼠腹外斜肌对结直肠扩张刺激放电反应显著增强,同等剂量下,对IBS大鼠的激动作用更明显(p0.05),而且在IBS大鼠该激动剂表现出明显的剂量依赖效应;Western blot结果显示,与正常大鼠相比较,IBS大鼠脊髓胸腰段和腰骶段P2X4受体显著表达(p0.05)及BDNF蛋白显著表达(p0.05);而且鞘内给予P2X4受体阻断剂后,BDNF在IBS大鼠脊髓表达的抑制程度显著高于正常大鼠(p0.05);免疫组化结果显示,与正常大鼠相比较,IBS大鼠脊髓背角Iba-1蛋白显著表达(p0.05),形态发生不同程度的肥大,而且鞘内给予P2X4受体阻断剂后,Iba-1在IBS大鼠脊髓背角的表达抑制程度显著高于正常大鼠,肥大状态被不同程度的减轻;荧光双标结果显示,IBS和正常大鼠P2X4受体和Iba-1蛋白在脊髓背角共表达。结论:脊髓小胶质细胞P2X4受体对慢性内脏痛中枢具有敏化作用,其机制可能是通过激活小胶质细胞释放脑神经营养因子导致了慢性内脏痛的中枢敏化。
[Abstract]:Aim: to investigate the central sensitization of microglia P2X4 receptor on the spinal cord of chronic visceral pain, and to provide a new target and therapeutic direction for the drug therapy of chronic visceral pain. Methods: Sprague-Dawley (SD) rats were born on the 3rd day of life and were given 3 hours maternal and infant separation at a fixed time every day to establish the model of chronic visceral pain in IBS rats with irritable bowel syndrome (IBS). The success of the establishment of IBS model was evaluated by detecting the discharge response of the ventral oblique muscle to colorectal dilatation, and the discharge amplitude of P2X4 receptor blocker 5-BDBD and the agonist C5-TDS before and after intrathecal administration of P2X4 receptor blocker 5-BDBD were recorded. The expression of P2X4 receptor and brain-neurotrophic factor BDNFin in rat spinal cord were detected by Western blot, and the activation status of microglia was observed by immunohistochemical method. The expression site of P2X4 receptor and Iba-1 protein in spinal dorsal horn of rats was observed by immunofluorescence double labeling method to confirm whether P2X4 receptor was expressed on microglia cells. Results: compared with normal rats, the response of external oblique abdominal muscles to colorectal dilatation was significantly enhanced in IBS rats, and 5-BDBD, a P2X4 receptor blocker, could inhibit the discharge response of IBS and normal rat external oblique muscles to colorectal dilatation, at the same dose, the same dose of P2X4 receptor antagonist 5-BDBD could inhibit the response of IBS and normal rats to colorectal dilatation. The blocking response to IBS was more obvious in IBS rats, and the antagonist showed a dose-dependent effect in IBS rats, and the responses of IBS and normal rat ventral oblique muscles to colorectal dilatation were significantly increased after intrathecal administration of P2X4 agonist C5-TDS. At the same dose, the excitatory effect on IBS rats was more obvious than that on IBS rats, and the agonist showed a significant dose-dependent effect in IBS rats. Compared with normal rats, the expression of P2X4 receptor and BDNF protein in thoracolumbar and lumbosacral segments of IBS rats were significantly higher than those in normal rats, and the inhibition degree of BDNF expression in IBS spinal cord after intrathecal administration of P2X4 receptor blocker was significantly higher than that in normal rats. The results of immunohistochemistry showed that, The expression of Iba-1 protein in the spinal dorsal horn of IBS rats was significantly higher than that of normal rats. The expression of Iba-1 in the spinal dorsal horn of IBS rats was significantly higher than that of normal rats. The expression of Iba-1 in the spinal dorsal horn of IBS rats was significantly higher than that of normal rats, and the expression of Iba-1 in the spinal dorsal horn of IBS rats was significantly inhibited by intrathecal administration of P2X4 receptor blocker. The hypertrophy was alleviated in varying degrees, and the results of fluorescence double labeling showed that P2X4 receptor and Iba-1 protein were co-expressed in the dorsal horn of spinal cord. Conclusion: P2X4 receptor of spinal microglia can sensitize the center of chronic visceral pain by activating microglia to release neurotrophic factor, which may lead to the central sensitization of chronic visceral pain.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R402

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