层粘连蛋白融合神经营养因子在喉返神经修复中的机制研究

We have reported that the recovery of laryngeal function is better than the pheripheral nerves in laryngeal transplatation.It is infered that reinnervation possess the capacity to regenerate but the reinnervation of muscles is often suboptimal and results in limited recovery of function. It is demonstrated that microenviroment is important for pheripheral nerves regeneration. Neurotrophic factors are playing critical roles in promoting reinnervation, such as BDNF and GDNF.After peripheral nerve injuries, laminin is significantly upregulated at the injury sites by Schwann cells and may foster asonal regeneration. Thus, laminin could be a suitable target for the delivery of exogenous neurotrophic factors to repair injuries.A laminin-bingding domain (LBD) was utilized to construct laminin-binding BDNF and laminin-binding GDNF.LBD-BDNF and LBD-GDNF could target accumulated laminin in the injury sites and exert targeting therapy of injured neurons. Inactivation of PTEN results in accelerating the reinnervation probably by the pathway of PI3K/Akt. To testify hypothesis above, we set up the different levels of rat model of recurrent laryngeal nerve injury, and discuss the function of LBD-BDNF and LBD-GDNF after applying in the sites of injury. Bidirectional regulating PTEN by gene transfection and siRNA, we observe the affect on reinnervation and PI3K/Akt. By testing the changes in function and electropsysiology of larynx and recurrent laryngeal nerves, histopathology of central and peripheral nervous muscle system, proteins of schwann cell. we will find a promising way to enhance the ability of peripheral nerves regeneration and functional recovery.

课题组前期研究发现，在喉移植中喉功能恢复要明显优于神经功能，单纯手术方式对神经修复作用有限，说明微环境对外周神经的再生是至关重要的；BDNF和GDNF是两个非常重要的神经营养因子，而外周神经损伤部位往往分泌大量laminin。以laminin为靶点，将神经营养因子与laminin结合位点融合，可能在神经再生中具有协同放大作用。构建生物胶原支架，保证横断神经有序生长。PTEN失活促进神经再生，该作用很可能是受PI3K/Akt信号途径调节。为验证以上假设，本项目建立不同类型大鼠喉返神经损伤模型，给予不同微环境作用，探讨对神经再生的促进能力。采用基因转染和siRNA，双向调节PTEN水平，观察对神经再生和PI3K/Akt信号途径影响。并从喉、喉返神经功能，中枢神经肌肉系统组织病理学，雪旺细胞蛋白质表达变化等方面探讨微环境对神经再生的作用和可能的信号途径，为提高外周神经再生能力和功能恢复奠定基础

课题组前期研究发现，微环境对外周神经的再生是至关重要的；BDNF和GDNF是两个非常重要的神经营养因子，而外周神经损伤部位往往分泌大量laminin。以laminin为靶点，将神经营养因子与laminin结合位点融合，可能在神经再生中具有协同放大作用。本研究构建LBD-BDNF、LBD-GDNF体外验证成功后，作用喉返神经挤压伤及横断伤动物模型， 发现两种合成物较之单纯的BDNF、GDNF有更好的促喉返神经修复的作用。且通过喉返神经损伤模型局部慢病毒转染敲除和过表达PTEN实验发现：在周围神经损伤中，通过抑制PTEN可激活 PI3K/AKT 信号途径加快神经修复。本研究完成既定目标，发表多篇学术论文，培养毕业博士2人。对临床喉返神经损伤治疗新药的研制具有指导作用，且对于喉返神经损伤修复的机制研究有了进一步的突破。