精确时控多种神经营养因子的雪旺细胞的构建及应用

Schwann cell (SC) serves as one of the most promising cells both for central and peripheral nerve repair. However, following SC transplantation, its ability to secrete neurotrophic factor (NTF) is reduced, which limits its role in nerve repair. How to improve the trophic role of SC and generate a microenvironment not only with multiple NTFs but also with controllable release kinetics is of great importance for successful nerve regeneration. This remains challenging worldwide. Previously, we found that c-Jun can co-regulate the expression of multiple NTFs in SCs. In this study, we attempt to use the newly developed tetracycline-regulatable Tet-On 3G system in a single lentiviral vector to control the expression of c-Jun. By administration or removal of doxycycline (Dox), a time-restricted, NTF-enriched regenerative microenvironment is established locally in the nerve injury site. Then, the Tet-on/c-Jun modified SC was transplanted to the peripheral nerve defect to investigate the effectiveness of time-restricted expression of multiple NTFs for nerve repair. In additon, the fate and safety of Tet-on/c-Jun modified SCs are traced and identified. The diffusion tensor imaging (DTI) technique was used to establish a "quantity-effect" relationship in which the length of the regenerated axon that enhanced by the up-regulated multiple NTFs and the final repair outcomes. Successful conduction of this study will be expected to construction of a new cell for nerve repair and enrichment of the theoretical system for delivery of NTFs in vivo.

雪旺细胞（SC）是神经损伤修复最具潜力的种子细胞之一。但是，SC移植后，分泌神经营养因子（NTF）能力下降，限制其作用的进一步发挥。如何提高SC营养活力，并根据再生阶段需求，精确时控多种NTFs释放，仍是尚未解决的世界性难题。本课题组前期研究发现c-Jun可同时调控SCs中多种NTFs表达。基于此，本项目拟利用可诱导型表达系统“Tet-On 3G系统”控制c-Jun以实现对SC内多种NTFs控制性表达。在此基础上，将Tet-on/c-Jun改造的SC应用于周围神经缺损，明确精确时控多种NTFs的SC对神经修复的有效性，并追踪移植细胞体内转归，明确其安全性。进一步应用临床无创的扩散张量成像（DTI）技术建立体内诱导多种NTFs促进神经再生长度与最终修复效果的“量-效”关系。本项目的成功开展可为神经损伤治疗提供新型种子细胞，还可以丰富体内因子递送理论体系，具有重要的理论意义和临床价值。

时控性递送多种神经营养因子（NTF）有望为神经再生提供更优的再生微环境，具有巨大的神经修复潜力。国际鲜有报道同时实现多种活性NTF时控性递送的方案。在本项研究中，我们以c-Jun作为“共同开关”，引入可诱导性基因表达系统（Tet-On 3G系统）作为“第二重开关”控制“共同开关c-Jun”，以实现对SC内多种NTFs控制性表达。体外研究表明，体外实验表明，Tet-On 3G系统可以通过添加或去除Dox来体外实现c-Jun控制性表达。同时，GDNF、NGF、BDNF和Artn在Tet-On 3G系统的控制下也表现出与c-Jun相同的可控性表达趋势。将Tet-On/c-Jun转染的SCs移植入大鼠坐骨神经缺损。通过饮水中口服Dox，在1周内诱导GDNF、NGF、BDNF和Artn的高表达。从饮水中去除Dox一周后，GDNF、NGF、BDNF和Artn的表达恢复至基础水平（与未转染SCs表达水平无显著差异）。体内诱导高表达NTFs可以显著提高再生轴突的数量及速度。双相因子控释方案组(NTFs上调3周，去除诱导使其恢复基础水平9周)，其运动及感觉神经再生数量均高于持续高表达组及未高表达组。更重要的是双相因子控释方案组电生理、肌肉萎缩评估、坐骨神经指数等功能恢复指标也均优于持续高表达组及未高表达组。发现诱导多种NTFs使神经再生穿过植段到达远端后去除诱导可获得最佳修复效果的“量-效”关系。本项目首次构建同时精确时控多种NTFs的雪旺细胞，为神经损伤治疗提供新型种子细胞；首次提出“单基因，多因子”因子递送方案，丰富了体内因子递送理论体系，具有重要的意义。本项目相关研究成果发表 SCI 论文2篇，中文核心期刊2篇，获授权发明专利2项，申报发明专利2项。培养博士研究生1名，硕士研究生2名。