深刺“环跳”在坐骨神经损伤修复中的抗凋亡作用及相关信号通路研究

Sciatic injury could be treated or symptom reduced by acupuncturing GB 30 point from the clinical researches. Early study has shown that deep stab GB 30 can promote neural electrical activity, alleviate the pathological damage of injury and we also found the differences of insertion depths could affect the expression of Bax and Bcl - 2 which were closely linked with apoptotis. However, the effects and its mechanism induced by the depth difference of insertions have not been sufficient studied yet. On the basis of previous work, we will establish the sciatic nerve injury model of rats again to observe the correlation between nerve cell apoptosis and acupuncture depth. Bcl-2、Bax、JNK、c-jun、caspase-3 of JNK signal path way would be detected to investigate the influence in protein and gene using the immunofluorescence, Western blot, rt-pcr technique of L4-5 dorsal root ganglion and injury site of sciatic nerve. For accurating insertion depths , small animal ultrasound instrument would be taken. Deep insertions must touch the nerve trunk under the detection of the instrument. The research would set non acupoint controls to make clear whether the different effects related to GB 30 point or organizational structure. This study will also use gene chip to screen signaling pathways related to cell growth and apoptosis for further exploring the effects and their mechanism of different depths from dorsal root ganglion L4-5 and injury site of sciatic nerve.

针刺环跳治疗坐骨神经损伤确有临床疗效，针刺深度不同疗效不同，但其具体作用机制尚不明确。本实验在前期工作基础上，建立坐骨神经损伤大鼠模型，利用小动物超声影像系统、电镜等进一步明确不同深度针刺环跳对坐骨神经损伤修复的疗效差异，并从细胞凋亡角度，运用免疫荧光、Western blot、RT-PCR等多项技术探讨针刺深度对L4-5背根神经节、损伤坐骨神经处JNK信号转导通路中Bcl-2、Bax、JNK1,2、c-jun、caspase-3蛋白和基因表达的影响。实验引进小动物超声仪，精确定位针刺深度，深刺以触及神经干为准，并设立非穴深刺对照，以明确环跳的穴位特异性作用。本研究还运用基因芯片技术筛查L4-5背根神经节、损伤坐骨神经处与针刺效应有关的其他细胞生长、凋亡相关信号通路的基因变化，为更加深入探讨针刺不同深度的效应机制奠定基础。

针刺治疗坐骨神经损伤确有临床疗效，针刺深度是影响针刺刺激量与针刺疗效的重要因素，针刺深度不同疗效不同，但其具体作用机制尚不明确。针刺的有效性深度的明确对针灸学术的规范化、标准化和国际交流具有重大意义。本项目制备坐骨神经损伤大鼠模型，利用“环跳”穴深层次解剖结构作为坐骨神经的特点,以不同的针刺深度进行针刺治疗,分两个层次(背根神经节L4-5、坐骨神经损伤处)检测损伤组织修复和神经细胞凋亡情况, 以JNK信号通路为切入点，运用多种分子生物学技术探讨针刺目标组织结构与针刺效应之间的关系。研究结果明确了①“环跳”穴的解剖结构和针刺深度，发现针刺可改善神经运动功能和传导功能，修复组织形态结构，通过抑制JNK信号通路的激活抑制坐骨神经和背根神经节神经细胞凋亡，深刺效果优于浅刺；②浅刺的差异基因表达主要集中于乙醛和二羧酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、甘油酯代谢等，与代谢、免疫调节、细胞紧密连接等方面有关，主要的信号通路为白细胞经内皮迁移、乙醛和二羧酸代谢，光传导等信号通路等，主要影响代谢反应、免疫过程。深刺的差异基因表达主要集中于瓦勒变性或抑制瓦勒变性过程、轴突引导、细胞间连接、吞噬细胞发育与形成雪旺细胞迁移雪旺细胞发育等与神经损伤、凋亡、发育、再生相关的过程。相关信号通路为细胞粘附分子信号通路、轴突引导信号通路、吞噬体信号通路，可对坐骨神经损伤修复起到良性的调节与治疗作用；③电针可抑制坐骨神经损伤大鼠凋亡与炎症反应，低频效果优于高频。上述发现为进一步探讨针刺不同深度的效应机制和针刺对神经损伤修复的作用机制亚信号通路筛选研究奠定了基础。

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