外源性Exosomes介导microRNA214在周围神经的轴浆转运及对脊神经节细胞存活和再生的影响

Exosomes is a nano membrane vesicles cells, can be used as an efficient carrier of miRNA. However, exogenous exosomes can be peripheral nerve endings uptake and transport is a problem urgently to be studied. We refined to exosomes in cultured DRG supernatants，this exosomes contains rich miRNA; also we found that miR-214 are differentially expressed in injury in DRG and miR-214 has an effect on cultured DRG neuron regeneration. Therefore, this project plans to use exosomes from mesenchymal stem cells (MSCs) transfected miR-214 modified lentiviral vector and the sciatic nerve injury model to study exosomes uptake and transportion in peripheral nerve, as well as the exosomes mediated miR-214 on regeneration of peripheral nerve. We look forward to clarify① rules and patterns of peripheral nerve uptake and transport of exogenous Exosomes; ②the potential significance of exosomes mediated miRNA in the treatment of peripheral neuropathy, injury and ③the role of miR-214 in sensory nerve regeneration in.The completion of the project is expected to provide a basis for the use of Exosomes as a vector for gene therapy

Exosomes是细胞分泌的纳米级膜性小泡，可作为一类高效的miRNA载体。但是，外源性Exosomes能否被周围神经末梢摄取并转运是亟待研究的问题。我们在培养的DRG上清液中提炼到Exosomes，此类Exosomes含有较丰富的miRNAs；也发现miR-214在损伤DRG中有差异性表达，miR-214对培养的DRG神经元再生有影响。因此，本项目拟利用miR-214修饰慢病毒，转染间充质多能干细胞（MSCs），获得MSCs来源的Exosomes。在坐骨神经损伤模型上，研究Exosomes在周围神经末梢的摄取及转运，以及经Exosomes介导的miR-214对周围神经再生的影响。期待初步阐明周围神经摄取转Exosomes的模式和规律；Exosomes介导miR-214在感觉神经再生中的作用。本项目的完成有望为利用Exosomes作为疾病基因治疗的载体提供基础。

Exosomes是细胞分泌的纳米级膜性小泡，可作为一类高效的miRNA载体。但是，外源性Exosomes能否被周围神经末梢摄取并转运是亟待研究的问题。本研究利用Exosomes的标志蛋白CD63耦联修饰带有绿色荧光的慢病毒，转染间充质干细胞（MSCs），获得能示踪Exosomes的MSCs，进而获得绿色荧光标记的MSCs来源的Exosomes。（1）经与模式细胞（BV2及SHSY5Y细胞）、离体DRG细胞共培养，以及坐骨神经切断模型上，研究了Exosomes在周围神经末梢的摄取及转运情况及其对周围神经再生的影响。初步阐明了周围神经摄取转Exosomes的模式和规律。（2）在大鼠坐骨神经压榨模型上，证明了MSCs来源的Exosomes具促周围神经再生的能力，外泌体与大鼠坐骨神经损伤的修复效果具有一定的量效关系，并验证了最优剂量。（3）miRNA21荷载致bMSC后，能更好地发挥促神经再生作用，这种作用可能与调节TIMP3、SPRY2信号通路相关。(4)外泌体还通过作用于DRG卫星细胞，发挥保护感觉神经元的作用，并促感觉神经元再生作用。