TGF-β1通过CXCR4/SDF-1α轴增强内源性神经干细胞迁移对喉返神经损伤后中枢疑核的修复机制

Patients with Recurrent laryngeal nerve (RLN) injury have a reduced quality of life and would experience dysphonia, aspiration and dysphagia by different degree. However, it is difficult to treat RLN paralysis for the loss of motor neurons in the nucleus ambiguus (NA) caused by RLN injury, which is one of the most important factors. It is presented in our previous research that neuroregeneration existed in the NA after RLN avulsion injury in rats. However, differentiated neurons failed to migrate into the NA. In this project, nutritional factor TGF-β1 would be implanted in the lateral ventricles, increasing the migratory ability of endogenous NSCs through CXCR4/SDF-1α axis to repair the neural circuits in the NA. In vitro, Western blot and Real-time PCR would be adopted for investigating whether and how TGF-β1 enhance NSCs migrating to the NA by CXCR4/SDF-1α axis; in vivo, immunofluorescence and laryngeal electromyography would be used to test and verify the repairing mechanism of TGF-β1. It is helpful for neural functional recovery and future study in the treatment of RLN injury.

喉返神经损伤会导致不同程度的发音及吞咽障碍，重者危及生命，外周神经损伤导致的中枢疑核运动神经元缺失，是导致喉返神经损伤治疗难度大的重要因素之一。申请人前期研究表明喉返神经撕脱伤后，中枢内源性神经干细胞具有迁移到受损疑核修复病变的潜能，但是分化为神经元的神经干细胞未能迁移到病灶。本课题拟用营养因子TGF-β1植入侧脑室，通过CXCR4/SDF-1α轴增强内源性神经干细胞的迁移能力来修复疑核神经回路，达到修复疑核病变的目的。利用Western blot和Real-time PCR的技术，从蛋白和基因水平探讨TGF-β1介导CXCR4/SDF-1α轴增强神经干细胞迁移的机制；采用免疫荧光和喉肌电图的方法，从动物水平验证TGF-β1通过CXCR4/SDF-1α轴介导内源性神经干细胞对疑核病变的修复作用，为喉返神经损伤的治疗提供新的思路。

喉返神经损伤会导致不同程度的发音及吞咽障碍，重者危及生命，外周神经损伤导致的中枢疑核运动神经元缺失，是导致喉返神经损伤治疗难度大的重要因素之一。本课题采用营养因子TGF-β1植入侧脑室，通过CXCR4/SDF-1α轴增强内源性神经干细胞的迁移能力来修复疑核神经回路，达到修复疑核病变的目的。实验结果：（1）体外实验部分，研究了TGF-β1在体外通过CXCR4/SDF-1α轴增强NSCs迁移能力的机制。利用Western blot和Real-time PCR的方法，从细胞水平和基因水平检测TGF-β1及其各通路抑制因子作用NSCs后的CXCR4表达水平，以及相关通路分子的磷酸化情况，从而探讨TGF-β1上调CXCR4的信号通路；利用Transwell迁移实验及Giemsa染色明确TGF-β1通过CXCR4/SDF-1α轴增强NSCs的迁移能力。（2）体内实验部分，研究了TGF-β1在RLN损伤后内源性NSCs迁移到受损同侧NA的修复作用。利用侧脑室立体定位注射CM-Dil标记室管膜区的内源性NSCs，通过激光共聚焦免疫荧光共定位结合层扫技术观察内源性NSCs在脑内的迁移情况；通过内源性NSCs对神经元、星形胶质细胞、少突胶质细胞及突触小泡蛋白等标记物的表达，来研究其分化方向；结合免疫组化染色的方法进一步观察疑核的修复情况，包括神经元细胞数目、突触形成及神经回路建立情况；采用透射电镜及HE染色，观察大鼠损伤-修复后的喉返神经轴突及髓鞘修复情况以及喉返神经的靶器官甲杓肌的变化情况。综上所述，TGF-β1可以通过CXCR4/SDF-1α轴介导内源性神经干细胞对疑核病变进行修复作用，为喉返神经损伤的治疗提供新的思路。