MicroRNA-30b靶向调节Sema3A对视神经损伤修复的作用

摘要

目的 对专MicroRNA-30b靶向调节Sema3A对视神经损伤的临床修复作用进行探究,进而进一步证实Sema3A是MicroRNA-30b的靶基因,明确MicroRNA-30b可以通过对Sema3A的调节来表达、抑制其信号通路,进而保护视网膜神经节细胞并加快轴突的生长,为视神经受损提供系的思路与目标靶点.方法 择取实验室现有的实验鼠进行SD视神经钳夹损伤模型,并分别采取qRT-PCR、Western blotting以及双荧光素酶报告基因系统对实验鼠健康及受损后,视网膜中MicroRNA-30b、Sema3A等指标的表达变化情况进行检测分析.向实验鼠损伤眼玻璃体腔内分别注射实验室现有的PBS、rAAV-miR-30b mimic、rAAV-miRNA NC以及rAAV-miR-30bInhibitor等制剂,进而判定损伤后不同时间节点内视网膜神经节细胞(RGCs)的存活率变化,以了解不同组别间视功能恢复情况的差异.最后,进行以Sema3A为靶目标的siRNA,分别在正常情况下体外培养RGCs和取目前抑制效果最佳的siRNA进行培养RGCs状态下检测Sema3A的表达量,以掌握MicroRNA-30b调控Sema3A的信号通路情况.进而判定MicroRNA-30b靶向调节Sema3A对视神经损伤修复中的具体作用.结果 在SD神经钳夹损伤模型中可知,实验鼠视网膜中MicroRNA-30b、Sema3A的表达均为先上升而后下降,一般在7 d时达最高峰,免疫组化检测显示Sema3A的表达一般发生于健康视网膜中的节细胞层与内核层中,而在视神经受损后两细胞层内阳细胞数量显著增多.同时,在实验鼠玻璃体内注射注射PBS、rAAV-miR-30b mimic、rAAV-miRNA NC以及rAAV-miR-30bInhibitor后受损四组RGCs存活率均有不同程度下降,且mimic组最高、inhibitor组最低.结论 实验表明,Sema3A为MicroRNA-30b的靶基因,在对视神经修复中具有显著的作用,能够有效提升视神经损伤后RGCs的存活率,进而促进视神经的修复.%Objective To explore the clinical effect of MicroRNA-30b in further specifically targeted regulation of Sema3A in repairing optic nerve injury, and to further confirm that the target gene of MicroRNA-30b.and it through the regulation of Sema3A expression, MicroRNA inhibit the signal path, thereby protect the retinal ganglion cells and accelerate the growth of axons in optic nerve in order to provide idea and damaged target.Methods Experimental rats were selected from the laboratory of this hospital for establishment of SD optic nerve crush injury model, and qRT-PCR, Western blotting and dual luciferase reporter gene system were taken on retina of healthy and damaged experimental rats after MicroRNA-30b, Sema3A and other indicators were analyzed.Experimental rats were injected intravitreal PBS, rAAV-miR-30b mimic, rAAV-miRNA NC and rAAV-miR-30b Inhibitor preparation, and then determine the survival rate in different time nodes within RGCs after injury, in the hope of understanding between different groups of visual function recovery differences.Finally, siRNA by targeting Sema3A, respectively under normal conditions in vitro RGCs and siRNA present the best inhibitory effect of Sema3A RGCs expression under the condition of culture, and then grasp the signal pathway of MicroRNA-30b regulation of Sema3A.Then the determination of MicroRNA-30b targeting Sema3A modulates the repair of optic nerve injury, the specific role of SD.Results The nerve injury rat models, MicroRNA-30b and Sema3A in retina index were increased firstly and then decreased, in general 7d reached the peak.The immunohistochemical study showed that the expression of Sema3A in ganglion cell layer occurred in healthy retina and kernel layer, and the number of positive cells in two cell layers increased significantly after the optic nerve damage.At the same time, the survival rates of rats after intravitreal injection of PBS, rAAV-miR-30b mimic RAAV-miRNA, NC and rAAV-miR-30b inhibitor, the ROCs survival rates of four group were decreased, and that in mimic group was the highest and inhabitor group was the lowest.Conclusion The experimental results showed that Sema3A is the target gene of MicroRNA-30b, it has a significant role in the repair of optic nerve, and it can effectively raise the survival rate of RGCs after optic nerve injury, and promote the repair of optic nerve.