嗅鞘细胞对P2X2/3受体介导神经病理痛的作用研究

The pain is a common symptom of most diseases, due to the complexity of the pain mechanism; its treatment has become a clinical problem. Transplantation of olfactory ensheathing cells (OECs) would promote regeneration of nerve, but there no relevant reports whether OECs can reduce neuropathic pain mediated by P2X2/3 receptors. In this study we intend to transplant OECs and microencapsulated OECs to the two sides of the injury sciatic nerve in pain models with chronic constriction injury. Pain behavioral changes were observed by the methods of behavioral observation. Sciatic nerve, dorsal root ganglion and spinal cord ultrastructural changes were observed under transmission electron microscope. The effects of OEC transplants on P2X2/3 receptor expression levels in the dorsal root ganglion and spinal cord were observed with immunohistochemical methods. Expression level of P2X2/3 receptor changes in dorsal root ganglion and spinal cord were determined by in situ hybridization combined with RT-PCR and western blot analysis. OEC transplants modulation and mechanism of currents activated by P2X2/3 receptor agonist in dorsal root ganglion and spinal cord gray matter dorsal horn neurons were studied with whole-cell patch-clamp technique. We determine whether the OECs impact the human P2X2/3 receptors by HEK293-hP2X2/3 cells electrophysiological studies. This project examines the role and mechanism of the OECs and microencapsulated OECs transplantation on the neuropathic pain and to explore new metholds of pain prevention.

疼痛是大多数疾病具有的共同症状，疼痛机理复杂，其治疗成为了临床上的难题。嗅鞘细胞能促进神经再生，能否减轻P2X2/3受体介导的神经病理痛，国内外未见相关报道。本实验拟将嗅鞘细胞和微囊化嗅鞘细胞移植至坐骨神经慢性压迫性损伤痛模型的坐骨神经周围；行为学方法观察痛模型行为变化；电镜观察坐骨神经和脊神经节等超微结构变化；免疫组化方法了解嗅鞘细胞对背根神经节和脊髓神经元P2X2/3受体表达水平的影响；原位杂交技术结合RT-PCR和蛋白质印迹分析等方法确定嗅鞘细胞移植对背根神经节和脊髓P2X2/3受体表达水平产生的变化；全细胞膜片钳技术研究嗅鞘细胞对背根神经节和脊髓灰质后角神经元P2X2/3受体激动剂激活电流的调制及机理。进行HEK293-hP2X2/3细胞的电生理研究确定嗅鞘细胞是否影响人类P2X2/3受体。通过本项目研究嗅鞘细胞和微囊化嗅鞘细胞移植对神经病理痛的作用及机理，为疼痛防治探索新方法。

疼痛是大多数疾病具有的共同症状，疼痛机制复杂，其治疗是临床上的难题。嗅鞘细胞能促进神经再生，能否减轻P2X2/3受体介导的神经病理痛国内外未见相关报道。本实验将嗅鞘细胞、微囊化嗅鞘细胞和嗅球组织分别移植至坐骨神经慢性压迫性损伤痛（CCI）模型的坐骨神经周围；行为学方法观察痛模型行为变化；电镜观察坐骨神经和脊神经节等超微结构变化；免疫组化方法检测嗅鞘细胞/组织对背根神经节和脊髓P2X2/3受体、P2X4受体、P2X7受体、NMDA、SP、NF-κB等表达水平的影响；原位杂交技术结合RT-PCR和蛋白质印迹分析等方法确定嗅鞘细胞/组织移植对背根神经节和脊髓P2X2/3受体、P2X4受体和P2X7受体等的表达水平变化；细胞膜片钳技术研究嗅鞘细胞对背根神经节和脊髓后灰质后角神经元P2X2/3受体激动剂激活电流的调制。进行HEK293-hP2X2/3细胞的电生理研究确定嗅鞘细胞是否影响人类P2X2/3受体。结果表明：嗅鞘细胞/组织移植及微囊化OECs移植均能减轻CCI大鼠的神经病理性疼痛症状，能抑制背根神经节及脊髓内P2X2/3受体、P2X4受体和P2X7受体等基因及蛋白表达水平。细胞膜片钳技术研究检测表明嗅鞘细胞抑制P2X2/3受体激动剂（ATP或 α,β-me ATP ）引起的背根神经节细胞膜去极化电位。HEK293-hP2X2/3细胞的电生理研究表明嗅鞘细胞对人类P2X2/3受体影响不太明显。本项目为神经病理痛的防治探索了一条新的方法，具有较好临床应用前景。