MiR-30b调控Sema3A在损伤视神经再生修复中的作用及机制研究

Nerve regeneration is limited after optic nerve injury mainly due to the inhibitory factors, that make the irreversible damage of visual function. Sema3A is an axon chemical exclusive content. There had evidence show that high expression of Sema3A is an important reason why the nerve regeneration fail, but the mechanism is still unknow.MiRNAs are known as an important way to regulate protein expression levels of target genes. We using bioinformatics website and Luciferase reporter assay found that miR-30 family, represented by miR-30b,may the most important reason of targeted regulating Sema3A. We plan to use Knock down, in situ hybridization technology and so on to make sure that miR-30b are targeted regulation molecular of Sema3A.We plan to use Knock down, in situ hybridization technology and so on to further verify that miR-30b are the targeted regulatory molecular of Sema3A, examine the biological function and molecular mechenisms of Sema3A regulated by miRNA and thus confirm that miR-30b promote the repairment of injuryed optic nerve by regulating Sema3A. The project should not only confirme the specific regulation miRNA of Sema3A, expand the miR-30b new biological function, but also provide a new idea and more effective targets for treatment of the optic nerve injury.

视神经损伤后再生能力有限，给伤者带来不可逆的视功能损害，其中抑制性因素是主要原因。Sema3A是一种轴突化学排斥物，已有证据显示视神经损伤时Sema3A高表达是抑制神经再生的重要原因，但其升高的机制尚不清楚。miRNA是基因表达调控的重要方式，我们根据生物信息学预测结果用荧光素酶报告基因活性检测发现以miR-30b为代表的miR-30家族可能是靶向调控Sema3A的一个重要因素。本项目拟采用Knock down、原位杂交等技术，进一步明确miR-30b是Sema3A的靶向调节分子，并通过miRNA调控Sema3A验证其生物学功能及其分子机制，从而证实增强miR-30b可以调控Sema3A达到促进视神经损伤修复以及视功能的恢复。本项目不仅可以确定调控Sema3A的特异miRNA，拓展miR-30b新的生物学功能，同时也为治疗视神经损伤提供一个新的思路和更有效的靶点。

作为中枢神经的一部分，视神经伤后恢复亦成为研究的热点和难点。众多的调控因素使视神经损伤后再生修复变得异常困难、复杂。Sema3A作为重要的轴突抑制物，在神经损伤后诱导生长锥塌陷，阻止神经修复，扮演着重要的调控角色。miRNAs在哺乳动物大脑、视网膜中特异性存在并且在其各个发育阶段动态表达，抑制Sema3A表达的miRNAs、为促进损伤视神经修复提供了新的思路。本项研究首先建立SD大鼠视神经钳夹损伤模型，发现视网膜中miR-30b表达先升高后下降，免疫组化检测发现，Sema3A表达在正常视网膜中内核层和节细胞层，伤后7d，节细胞层和内核层的阳性细胞明显增多。miR-30b与Sema3A的结合位点为UGUUUACA，转染miR-30b到体外培养RGCs，发现miR-30b mimic组中Sema3A、NRP1、PlexA1、p-p38MAPK和active caspase-3表达量最低，而miR-30b inhibit组最高；流式细胞仪检测发现miR-30b mimic组RGCs凋亡率最低，miR-30b inhibit组最高，RGCs轴突长度miR-30b mimic组最长，miR-30b inhibit组最短。大鼠视神经钳夹伤后，玻璃体腔内分别注射rAAV-miR-30b mimic、rAAV-miR-30b inhibitor、rAAV-miRNA NC和 PBS，miR-30b可通过抑制Sema3A表达改变NRP1、PlexA1的水平，促进RGCs轴突生长，并加快视神经损伤后视功能恢复，这可能是miR-30b促进损伤后视神经修复的主要分子机制。

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