诱导表达RhoA shRNA对RhoA信号的影响及其轴突再生作用研究

The activation of RhoA by a variety of inhibitory molecules leads to the failure of central nerve regeneration. Blocking the RhoA signal is an important strategy to promote nerve regeneration. Our previous studies have found that the RhoA shRNA expressed by the vector can specifically, efficiently and long-time block the RhoA signal and thus promote axon regeneration. However, the pathological RhoA signal activated by nerve injury has the characteristics of dynamic change. It is not clear how to eliminate the dynamic change of pathological RhoA signal. This project suggests that the expression of RhoA shRNAs can be induced by inductive expression vector according to the activation degree and change law of pathological RhoA signal, and the dynamic change of pathological RhoA signal can be eliminated at the right time, which can further promote nerve regeneration. In order to verify this hypothesis, we will use inducible AAV2-RhoA shRNA expression vector. In the model of cell, tissue and optic nerve injury, the expression of RhoA shRNA was induced. The changes of pathological RhoA signal and the effect of neurite growth / axon regeneration were observed. The purpose of this project is to explore a new method to promote nerve regeneration and to find new treatment for axon injury diseases in the central nervous system.

多种神经再生抑制分子激活RhoA导致中枢神经再生失败。阻断RhoA信号是促进神经再生的重要策略。我们前期研究发现，载体表达的RhoA shRNA可特异、高效、长期阻断RhoA信号，促进中枢神经再生。然而，神经损伤激活的病理性RhoA信号具有动态变化的特点。如何消除动态变化的病理性RhoA信号，目前不清楚。本项目认为，应用诱导型表达载体，针对病理性RhoA信号的激活程度及变化规律，有计划地诱导表达RhoA shRNA，适时消除动态变化的病理性RhoA信号，可进一步促进神经再生。为验证此假说，我们将应用诱导型AAV2-RhoA shRNA表达载体，在细胞、组织及视神经损伤模型上，诱导表达RhoA shRNA。通过观察病理性RhoA信号的变化及神经突起生长/轴突再生效应，明确诱导表达RhoA shRNA的神经再生促进作用。本项目探索促进神经再生的新方法，为治疗中枢神经轴突损伤性疾病寻找新的手段。

创伤、缺血等疾病损伤中枢神经轴突，引起神经元与其靶细胞的连接中断，导致神经功能障碍。由于中枢神经轴突再生十分困难。促进神经再生，使断裂的轴突与靶细胞重新建立连接，是治疗神经轴突损伤性疾病的关键环节。遗憾的是，时至今日临床仍无理想的神经再生促进措施。既往研究发现，多种神经再生抑制分子激活RhoA导致中枢神经再生失败。阻断RhoA信号是促进神经再生的重要策略。我们前期研究发现，载体表达的RhoA shRNA可特异、高效、长期阻断RhoA信号，促进中枢神经再生。然而，神经损伤激活的病理性RhoA信号具有动态变化的特点。如何消除动态变化的病理性RhoA信号，目前未见研究。本项目应用诱导型表达载体，针对病理性RhoA信号的激活程度及变化规律，有计划地诱导表达RhoA shRNA，适时消除动态变化的病理性RhoA信号，进一步促进神经再生。本研究应用诱导型LV-RhoA shRNA表达载体，在细胞、组织及神经损伤模型上，诱导表达RhoA shRNA。通过观察病理性RhoA信号的变化及神经突起生长/轴突再生效应，明确了诱导表达RhoA shRNA的神经再生促进作用。研究发现，诱导型LV-RhoA shRNA表达载体比常规型载体对PC12细胞RhoA信号的阻断作用更好、更适度；诱导型LV-RhoA shRNA表达载体能更好地促进PC12细胞神经突起生长；诱导型LV-RhoA shRNA表达载体比常规型载体对RGC RhoA信号的阻断作用更好、更适度，诱导型LV-RhoA shRNA表达载体能更好地促进RGC神经突起生长，其效果显著优于常规方法；诱导型LV-RhoA shRNA表达载体比常规型载体对神经细胞 RhoA信号的阻断作用更好、更适度；诱导型LV-RhoA shRNA表达载体能更好地促进神经细胞轴突再生，其效果显著优于常规方法。本项目探索了促进神经再生的新方法，为治疗中枢神经轴突损伤性疾病提供了新的实验依据及启示。