移植微囊化雪旺细胞对P2X2/3受体介导神经病理性疼痛的作用研究

Schwann cells are the special target which are used to treat nerve injury and alleviate neuropathic pain. Our previous studies have shown that transplantation of the free and microencapsulated olfactory ensheathing cells which similar to Schwann cells function and form can relieve neuropathic pain. There are no related reports about whether transplantation of microencapsulated Schwann cells can alleviate neuropathic pain mediated by P2X2/3 receptor. In the present study, we microencapsulated Schwann cells in alginic acid, and transplanted free and microencapsulated Schwann cells into the region surrounding the injured sciatic nerve in rat models of chronic constriction injury. Mechanical withdrawal threshold and thermal withdrawal latency were measured to detect the changes of neuropathic pain behavior in CCI rats. The ultrastructural changes of the injured sciatic nerve and spinal cord were observed by electron microscopy. Effects of Schwann cells on the levels of P2X2/3 receptor in dorsal root ganglion and spinal cord were detected by immunohistochemical method. We determined P2X2/3 receptor expressions in the dorsal root ganglion and spinal cord with in situ hybridization technique, with RT-PCR , Western blotting analysis and other methods. Using whole cell patch clamp technique and laser confocal technique to observe the fast regulation function of olfactory ensheathing cells in cultured dorsal root ganglion neurons ATP activation current and intracellular calcium ion concentration increases caused by ATP, and discusses the mechanism of this regulation.We determine whether the SCs impact the human P2X2/3 receptors by HEK293-hP2X2/3 cells electrophysiological studies.Through this project to investigate whether it can alleviate the pathological pain caused by P2X2/3 receptor stimulation by transplantation of microencapsulated Schwann cells and to explore new methods for prevention and treatment of neuropathic pain.

雪旺细胞（SCs）是治疗神经损伤与减轻神经病理性疼痛的特殊治疗靶点。我们前期研究表明与其功能形态相似的嗅鞘细胞能减轻神经病理性疼痛，但关于移植微囊化SCs对疼痛影响及机制方面的研究少见。本项目拟将微囊化雪旺细胞移植至大鼠痛模型受压迫的坐骨神经周围；观察痛模型的行为学变化；电镜观察损伤侧坐骨神经及脊神经节的超微结构变化；免疫组化方法检测背根神经节和脊髓内P2X2/3受体水平的变化；原位杂交技术结合RT-PCR和蛋白质印迹分析等方法检测P2X2/3受体表达水平变化；采用全细胞膜片钳技术及激光共聚焦技术观察雪旺细胞对培养的背根神经节神经元ATP激活电流及ATP所致的细胞内钙离子浓度升高的快速调控作用，并对这种调节作用的机制进行探讨。电生理研究微囊化SCs是否影响人类P2X2/3受体。探讨微囊化SCs移植对神经病理性疼痛的影响及机制，扩大SCs的应用范围，为临床上减轻患者的疼痛提供新的方法及思路。

雪旺细胞（SCs）是治疗神经损伤与减轻神经病理性疼痛的特殊治疗靶点。本项目将微囊化雪旺细胞移植至坐骨神经痛大鼠模型受压迫损伤（CCI）的坐骨神经周围；观察痛模型的行为学变化；电镜观察损伤侧坐骨神经及脊神经节的超微结构变化；免疫组化方法检测背根神经节和脊髓内P2X2/3受体水平变化；原位杂交技术结合RT-PCR和蛋白质印迹分析等方法检测P2X2/3受体表达水平变化；采用全细胞膜片钳技术及激光共聚焦技术观察雪旺细胞对培养的背根神经节神经元ATP激活电流及ATP所致的细胞内钙离子浓度升高的快速调控作用，并对这种调节作用的机制进行探讨。电生理研究微囊化SCs是否影响人类P2X2/3受体。结果：1.行为学变化观察表明：痛阈比较：于术前和术后2、4、6、8、10、14d测定机械缩足反射阈值（MWT）和热缩足反射潜伏期（TWL），CCI组术后MWT、TWL与Sham组相比明显降低；而通过微囊化雪旺细胞移植后大鼠MWT和TWL与CCI组比较明显升高。表明雪旺细胞移植可以减轻坐骨神经压迫损伤所致的神经病理性疼痛。2.电镜检测表明移植微囊化雪旺细胞可以促进受损坐骨神经形态结构的修复。3.CCI大鼠模型脊神经节P2X2/3mRNA及其蛋白含量较假手术组（Sham）高。微囊化雪旺细胞移植后CCI大鼠神经病理性疼痛减轻，脊神经节的P2X2/3mRNA及蛋白含量与神经病理痛组比较下降。4.细胞膜片钳技术研究检测表明雪旺细胞抑制P2X2/3受体激动剂（ATP或α,β-meATP）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雪旺细胞细胞对人类P2X2/3受体有一定影响。科学意义：探讨了微囊化SCs移植对神经病理性疼痛的影响及机制，扩大SCs的应用范围，为临床上减轻患者的疼痛提供新的方法及思路，具有较好临床应用前景。