MicroRNA-222和MicroRNA-338双慢病毒序贯表达系统协同促进神经损伤修复的作用及机理研究

Application of a variety of means could promote regeneration of injured sciatic nerve for different degrees, but new nerve fibers generally have low myelinations, and ultimately affect recovery of nerve function. Preliminary experimental research showed that, miRNA-222 could promote the proliferation of Schwann cells (SCs), miRNA338 helped SCs form myelin sheath. Accordingly, we propose the assumption that in the repair of peripheral nerve injury, the sequential expression of miRNA-222 and miRNA-338 should be regulated. The initial expression of miRNA-222 promotes SCS proliferation, accelerating the formation of Büngner band, to guide and promote axonal extension; expression of miRNA-338 in the repairing period can regulate myelination factor of SCs, and promote new axonal myelination. In the base of improving the in vitro experiments, this project aims to further adopt in vivo sciatic nerve injury model of rats, using double lentiviral system to regulate miRNA-222 and miRNA-338 sequential expression, and assess the synergistic effect on the sciatic nerve injury. And on this basis to further elucidate synergistic effect mechanism of miRNA-222 and miRNA-338 on repairing of sciatic nerve injury.

应用多种手段都可不同程度地促进损伤神经的再生，但新生轴突髓鞘化程度较低，主要是由于新生施万细胞（SCs）形成的髓鞘细薄，最终影响再生神经传导功能的恢复。前期实验研究表明，miRNA-222能够促进SCs增殖，miRNA338有助于SCs形成髓鞘。据此，我们提出这样的假设：在损伤神经修复过程中，序贯表达两个调控基因miRNA-222和miRNA-338，即损伤初期先表达miRNA-222促进SCs增殖，加速Büngner带形成，引导轴突的延伸；经过特定的时间窗再表达miRNA-338调控SCs的髓鞘化因子，促进新生神经轴突的髓鞘化。本项目在进一步完善体外细胞学实验基础上，拟在大鼠体内坐骨神经损伤模型中采用双慢病毒系统调控miRNA-222及miRNA-338序贯表达，评估其对损伤坐骨神经修复的协同促进作用，并进一步阐明miRNA-222和miRNA-338协同促进坐骨神经损伤修复的作用机理。

优化自体雪旺细胞(SCs)在神经修复不同阶段的功能是本项目关注的问题。本项目及既往的研究表明miR-221-3p增强了SCs的增殖和迁移，miR-338-3p促进了SCs的髓鞘化。在体实验中以导管桥接大鼠离断坐骨神经构建神经再生室模型，术中将携带miRNA-221常规慢病毒载体和强力霉素诱导的携带miRNA-338-Tet on的慢病毒载体系统共同注入神经断端导管腔内来有序调控内源性施万细胞的生物学功能，即实现miRNA-221-3p过表达促使新生神经穿过神经断端空隙后能立刻启动miR-338-3p的表达促进施万细胞髓鞘化。在体评估有序调节内源性施万细胞的生物学功能对损伤坐骨神经再生及功能恢复的协同促进作用，并进一步通过nanopore全转录组基因测序、qPCR和双荧光素酶实验确定miR-221-3p和miR-338-3p的作用靶基因分别是Cdkn1b和Nrp1，细胞实验证实miR-221-3p通过下调Cdkn1b促进SCs的增殖和迁移，miR-338-3p通过下调Nrp1促进SCs的髓鞘化。总之本项目利用miRNA-221和miRNA-338双慢病毒系统作为工具，通过体内外实验来阐明有序调控施万细胞的功能属性促进坐骨神经损伤修复的作用机理。这是首次通过有序调节SCs生物学特性来促进神经再生的研究，为周围神经损伤的治疗建立了新的概念和模型。本项目的完成，不仅为整个周围神经损伤的修复研究提供新的思路和方法，而且将会对临床上治疗坐骨神经损伤具有直接的指导意义。.结论：.1．miR-221-3p和miR-338-3p参与损伤坐骨神经的修复。miR-221-3p能够促进施万细胞增殖和迁移。miR-338-3p增加施万细胞髓鞘化水平。.2．体内序贯表达miR-221-3p和miR-338-3p促进损伤坐骨神经再生及功能恢复。.3．Cdkn1b和Nrp1分别是miR-221-3p和miR-338-3p的靶基因。miR-221-3p通过下调Cdkn1b促进SCs的增殖和迁移。miR-338-3p通过下调Nrp1促进SCs的髓鞘化。