新型纳米荧光粒子对周围神经修复早期疗效的活体示踪研究

Early in vivo observation of histologic changes of peripheral nerve injury and regeneration is crucial for predicting functional outcomes, however, current clinical methods can't satisfy this requirment. Neuroanatomical tracing technique can directly measure the level of peripheral nerve regeneration and estimate functional recovery of peripheral nerve. However, traditional neural tracers have several limitations, such as time-consuming staining procedures, weak fluorescence signal and rapid quenching. Therefore, we have been trying to create a novel fluorescent nano-neural tracing method. The novel carbon dots-based fluroescent neural tracers possess high photoluminescent intensity and good optical stability, but there are also several drawbacks due to short wavelength, including strong auto-fluorescence and low signal-to-noise ratio. We plan to create a novel in vivo neural tracing method based on near infrared quantum dots . In this way, we can evaluate the outcomes of peripheral nerve repair and functional recovery early and precisely. Moreover, we use double or multiple tracing methods based on multi-color quantum dots to study the mechanisms of structural and functional plasticity of peripheral nerve regeneration. Last, we change the ligands to synthesize different neural tracers with different features, such as antegrade tracing or trans-synaptic tracing, in order to expanding the scope of applications. In conclusion, we are going to synthesize a novel, inexpensive, nontoxic neural tracer and establish an in vivo method measuring peripheral nerve regeneration early and precisely. This project will be significant of deeply understanding of the whole process in peripheral nerve regeneration and functional recovery, improving the entire therapeutic effects of peripheral nerve injury.

早期活体观测周围神经损伤再生的组织学变化对于判断神经修复疗效及预测功能转归具有重要意义，而目前临床检查尚无法达到要求。神经示踪技术可直观检测神经再生水平，判断神经功能恢复的程度，但传统示踪技术操作复杂费时，且荧光强度低、易光漂白。因此，本课题组一直致力于研发新型纳米荧光神经示踪技术，最新合成碳点示踪剂荧光强且稳定，但其波长短，故生物组织自发荧光较强，局限于组织切片检查。本课题拟利用新型量子点创立活体荧光神经示踪技术，直观、准确地判断周围神经损伤修复早期疗效以及预测功能转归；并应用多色量子点对不同功能神经移位术进行双重或多重标记，揭示周围神经再生过程中结构与功能的重塑机制；改变亲神经性配体创制不同功能神经示踪剂，研究顺行或跨突触示踪的可行性，拓展其应用范围。通过本研究，可望为周围神经修复早期疗效评价提供一个活体观测方法，对深入理解周围神经再生机制，提高周围神经损伤的总体治疗水平具有重要意义。

周围神经损伤是临床常见病，也是疑难病，具有较高致残率。目前，周围神经修复临床评价方法均基于轴突靶器官重建联系而建立，无法准确、直观、及时地判断神经修复效果及预测功能转归。神经示踪技术是神经损伤后轴突再生检测的金标准，能直接反映神经再生的组织学变化。荧光纳米粒子具有独特的光学性质和良好的光稳定性，本组前期构建基于轴浆运输的新型荧光纳米神经示踪体系（FNNTs），但第一代FNNTs（碳纳米点-霍乱毒素B亚基，CTB-CDs）发射波长较短（435nm，蓝色）所致自发荧光强、信噪比较低，活体成像应用受限。由此，本课题围绕周围神经损伤再生早期检测与早期干预开展三方面工作：一、采用不同荧光纳米粒子及改变亲神经性配体，制备多功能第二代FNNTs，包括具有顺行示踪特性的聚合物碳点-生物素葡聚糖胺（BDA–CPDs）以及长时稳定的金纳米点-霍乱毒素B亚基（AuNDs-CTB），其优点是生物安全性好、荧光稳定性强、发射波长较长（630nm，红色）、自发荧光较低且信噪比较高，缺点是荧光组织穿透性较差、活体成像应用效果一般；二、构筑基于共价有机框架（COFs）和金属有机框架（MOFs）的多功能生物传感器，在复杂细胞微环境响应及检测、组织局部催化反应等方面开展研究，可望实现周围神经损伤后再生微环境的超敏检测；三、研究周围神经损伤相关轴突导向机制，发现轴突再生排斥因子（Sema3A）及其调节因子cdc42在坐骨神经损伤再生和交感神经分布中起关键调控作用。本课题发表相关SCI文章19篇，最高影响因子9.5，申请国家发明专利1项，获得第66批中国博士后面上项目一等资助，并基于此，负责人前往美国斯坦福大学进行近红外二区活体成像相关访问博士后研究。本课题为周围神经损伤再生研究提供先进技术，也为临床应用奠定实验基础，同时对深入理解周围神经再生机制，提高周围神经损伤的总体治疗水平具有重要意义。