电针通过NT-3介导炎症调节以促进移植的MSC源性神经网络在损伤脊髓内存活及功能性整合的研究

We successfully constructed the mesenchymal stem cells (MSC)-derived neural network with the function of synaptic transmission in vitro. Although MSC-derived neural network transplanted into the injured spinal cord can reconstruct partly the neural circuits of spinal cord, the continuous inflammatory microenvironment in the injured spinal cord severely interfered with the long-term survival and integration of transplanted neural network cells with the host neural circuits. Our previous results demonstrated that Governor Vessel electron-acupuncture (EA) could promote the neurotrophin-3 (NT-3) secretion of spinal cord tissue to inhibit inflammation and promote the survival of transplanted stem cells in the spinal cord. However, It is unclear how EA regulates inflammation mediated by NT-3 to promote the survival and integration of transplanted neural network with the spinal cord. Therefore, the hypothesis of this study is that: EA simulating meningeal branch of spinal nerve can increase the NT-3 secretion of injured spinal cord tissue, NT-3 mediates local immune cells to secrete anti-inflammatory factor IL-4 which induced proinflammatory cells to transform into anti-inflammatory cells in both sides tissue of transplanted neural network, inhibit inflammation and improve spinal cord injury microenvironment, protect the survival of the MSC neural network cells, and promote better integration of MSC-derived neural network with the host neural circuits and enhance the effect of repairing spinal cord injury. On the basis of our previous work, this study will utilize optogenetics technology to further precisely prove the EA afferent signal pathway which promote the NT-3 secretion of spinal cord and the mechanism of EA anti-inflammation. The study will provide the theory and experimental evidence for clinically applying Governor Vessel electroacupuncture combined tissue engineering neural network transplantation therapy the patient with spinal cord injury.

我们成功构建了具有突触传递功能的间充质干细胞(MSC)源性神经网络，将其移植到脊髓损伤处能部分重建神经环路。但损伤脊髓内持续的炎症反应影响移植神经网络的长期存活及与宿主神经环路的整合。现已证实，督脉电针可通过增强脊髓分泌NT-3，抑制炎症促进移植细胞存活。然而，督脉电针是如何通过NT-3介导炎症调控，促进移植神经网存活的机制尚不明确。因此，本项研究假设：督脉电针刺激脊膜支引起受损脊髓组织分泌NT-3，通过NT-3介导局部免疫细胞分泌抗炎因子IL-4，诱导位于移植神经网络两侧宿主组织内的促炎细胞向抗炎细胞转化，抑制炎症反应，改善脊髓损伤区微环境，保护MSC源性神经网络细胞并促进其与宿主脊髓神经环路更好地整合，提高修复脊髓损伤的效果。本研究将在前期工作基础上利用光遗传学等技术精准探明督脉电针促进脊髓NT-3分泌及其抗炎机制，为临床应用督脉电针联合组织工程神经网络修复脊髓损伤提供理论和实验依据。

脊髓损伤后炎症反应神经环路重建是未解决的关键科学问题，原因是受损伤脊髓微环境难以让内源性神经元修复神经环路。联合应用能够改善脊髓组织微环境的督脉电针与干细胞源性神经网络组织移植是解决这问题的关键策略。本研究已证实：电针督脉经穴可通过刺激脊膜支促进背根神经节神经元的传入神经末梢在脊髓内释放降钙素基因相关肽增多，作用于表达降钙素基因相关肽受体的脊髓神经元，激动其细胞膜上的L-型电压门控钙离子通道，通过上调钙/钙调蛋白-依赖性蛋白激酶通路促进神经元合成和分泌NT-3。通过体内外实验进一步证实，督脉电针通过增加NT-3的水平，作用于表达其受体TrkC的炎症细胞，通过激活IL-4/IL-4R/STAT6信号通路诱导损伤激活的M1型巨噬细胞向M2型巨噬细胞极化，从而下调促炎因子的分泌和上调抗炎因子的分泌，减轻脊髓损伤后的炎症反应，进而改善受损伤脊髓组织的微环境有利于细胞存活。在此研究基础上，督脉电针可以通过提高全横断脊髓损伤组织内NT-3的水平，促进移植入脊髓损伤区的MSC源性神经网络组织细胞存活，并维持其分化为干细胞源性神经元样细胞的特征，保持干细胞源性神经元样细胞之间形成的突触样结构，促进部分干细胞源性神经元样细胞往脊髓损伤/移植区尾端迁移，并与宿主脊髓神经元形成更多接触，促使移植的干细胞源性神经网络组织与宿主脊髓神经环路整合，从而改善全横断脊髓损伤大鼠的瘫痪肢体自主运动功能。本项研究从督脉经穴电针、内源性神经营养因子表达、移植的干细胞源性神经元与宿主脊髓神经环路整合修复脊髓损伤这主干线，进一步阐明了传统中医在应用针灸(或电针) 治疗脊髓损伤时均要首选督脉穴的机理。同时，为将来临床上解释移植干细胞源性神经网络组织的脊髓损伤患者在手术后，如果联合应用督脉穴电针治疗可以得到更好治疗效果的道理。所以，本项研究既有重要的科学意义，又有潜在的临床转化前景。

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