基于HIF-1a/VEGF信号通路探讨电针刺激对脊髓损伤神经再生微环境调控机制研究

To rebuild the oxygen microenvironment and activation of endogenous neural stem cell after spinal cord injury (SCI) are key factors in promoting nerve regeneration. HIF-1α/VEGF is the central signaling pathway in regulation of ischemia and hypoxia, but the mechanism for oxygen microenvironment reconstruction is still poorly understood after SCI. Our group preliminary use electroacupuncture to treat SCI based on the theory of the "rule a trophy was given the Yangming" and "reconcile Qi and blood" of traditional Chinese medicine (TCM), and got an excellent clinical efficacy. Therefore, we speculate "electroacupuncture could improve spinal cord blood oxygen microenvironment to promote nerve regeneration by regulating HIF-1α/VEGF signaling pathway ". In order to clarify this, At first,we technology to observed functional repair of nerve, the promoting nerve regeneration in the spinal cord injury in rats, to clarify the mechanisms of electroacupuncture to promote nerve regeneration based on by regulating HIF-1α/VEGF signaling pathway, then apply Cell pathology and molecular biological technology to analyze related rembers of HIF-1α/VEGF signal pathway, and to speculate the mechanisms of its regulating oxygen microenvironment.To detect HIF-1α/VEGF signaling pathways associated protein after electroacupuncture，and to observe functional repair of nerve, the promoting nerve regeneration in the spinal cord injury in rats, to clarify the mechanisms of electroacupuncture to promote nerve regeneration based on by regulating HIF-1α/VEGF signaling pathway and inflammatory cytokines,nerve growth factors，apoptosis-related molecule and activation of endogenous neural stem cell which are important to further explore more effective electroacupuncture treatment program for SCI.

脊髓损伤(SCI) 后血氧微环境构建及内源性神经干细胞的激活是决定神经再生的关键因素，HIF-1a/VEGF是调控缺血缺氧的重要信号通路，但对其如何参与SCI血氧微环境重建机理尚不清楚。本课题组前期依据中医"治萎独取阳明"、"调理气血"的理论，使用电针治疗SCI取得满意临床疗效。据此提出了"电针可通过干预HIF-1a/VEGF信号转导，改善脊髓血氧微环境，促进神经再生"的假说，拟通过建立大鼠SCI模型，应用基因芯片等技术观察HIF-1a/VEGF信号通路对脊髓血氧微环境构建的调控作用；应用细胞病理及分子生物学相关技术，观察电针对脊髓功能修复、神经再生、HIF-1a/VEGF信号通路相关分子、炎症因子、内源神经生长因子、及凋亡相关分子表达及内源性神经干细胞的激活的影响，从而阐明电针调控HIF-1a/VEGF信号通路促进神经再生治疗脊髓损伤的机理。

脊髓损伤(SCI) 后血氧微环境构建及内源性神经干细胞的激活是决定神经再生的关键因素，HIF-1a/VEGF是调控缺血缺氧的重要信号通路，但SCI血氧微环境重建机理尚不清楚。本课题提出“电针可通过干预HIF-1a/VEGF信号转导，改善脊髓血氧微环境，促进神经再生”的假说，通过建立大鼠SCI模型，将120只脊髓损伤大鼠模型随机分为阿是穴电针组、足阳明胃经电针组和空白对照组3组。观察7d、14d、21d、28d、35d时间节点，通过神经功能评价，免疫组织化学染色观察HIF-1ａ/VEGF信号转导通路相关蛋白HIF-1ａ、VEGF、PKA、synapsin I、CaMPKⅡ和BDNF、c-fos、c-jun表达。用qRT-PCR技术检测HIF-1ａ/VEGF信号转导通路相关mRNA表达。用Western blot技术检测HIF-1ａ/VEGF信号转导通路相关蛋白HIF-1ａ、VEGF、PKA、synapsin I、CaMPKⅡ和BDNF、c-fos、c-jun表达。结果①电针刺激通过调控HIF-1a/VEGF信号通路相关分子蛋白的表达对脊髓损伤大鼠产生治疗作用。②电针刺激可以上调HIF-1a/VEGF信号通路相关因子HIF-1a、VDGF、PKA、Synapsin I、BDNF基因和相应蛋白表达，抑制CaMKII 、C-fos和C-Jun基因和相应蛋白表达实现对脊髓损伤的治疗作用。③电针刺激可以促进神经营养因子并抑制炎症因子的表达，恢复神经元坏死结构，促进内源神经干细胞大量增殖，并替代受损神经细胞，抑制神经干细胞向胶质细胞分化，减少纤维瘢痕的形成，促进脊髓损伤的修复。实验数据表明：脊髓损伤大鼠下肢神经功能评分提示脊髓损伤后大鼠下肢运动功能存在可逆性的修复现象，可能是中枢神经系统自我修复机制的一部分，同时电针干预对脊髓损伤后大鼠的下肢运动功能肌力恢复在时间和效果上优于未经干预的脊髓损伤大鼠。证明电针可改变神经干细胞的微环境，影响一些重要的信号通路的变化，促进脊髓损伤大鼠下肢运动功能的恢复，提高其行为学评分。为脊髓损伤的后续治疗提供了一种新思路，为脊髓损伤再生修复的基础研究及临床转化研究提供参考，同时也对电针促进神经功能恢复作用的现代医学实验内涵提供了佐证。提示电针干预是治疗脊髓损伤的有效途径之一。