基于miR-20a-5p调控RGMa的相关通路在颞叶癫痫中的作用及机制研究

Temporal lobe epilepsy is the most common type of refractory epilepsy. Its nosogenesis is deemed to associate with mossy fiber sprouting of hippocampus. Repulsive guidance molecular a (RGMa) is a novel type of axon guidance and axon growth inhibitory factor，and plays an role in axon regeneration of several nervous system diseases. Bioinformatics predicts that RGMa is one of target genes of miR-20a-5p. And our previous study also found that RGMa is down-regulated while miR-20a-5p is up-regulated. It indicates that miR-20a-5p regulates expression of RGMa. Moreover, Phosphorylated FAK(Tyr397) and Ras protein are also raised in pentylenetetrazole kindling epileptic model. They are related to mossy fiber sprouting and temporal lobe epilepsy, but profound mechanisms remained to be unknown. This subject intends to adopting gene transfection, antisense oligonucleotide technology et al, combined with recently developed Designer Receptors Exclusively Activated by Designer Drugs to active exclusively granular cell, to explore relationship between activation of granule cells and mossy fiber sprouting and temporal lobe epilepsy, and influence of activated granule cells on RGMa related pathway-based miR-20a-5p by detecting the primary indexes of RGMa related pathway-based miR-20a-5p and electrophysiology of granule cells from origin of mossy fiber sprouting. Finally, we will clarify the role of RGMa related pathway-based miR-20a-5p in the mossy fiber sprouting and temporal lobe epilepsy. This will help to find an new target of treatment of temporal lobe epilepsy, and have an potential prospect of clinical application.

颞叶癫痫(TLE)是最常见的难治性癫痫，研究认为发病机制与海马苔藓纤维出芽(MFS)有关。排斥性导向分子a(RGMa)是一种新型的轴突导向和生长抑制因子，与多种神经系统疾病轴突再生相关。生物信息学预测RGMa是miR-20a-5p靶基因，我们研究也发现，在戊四氮点燃模型中，miR-20a-5p表达上调，RGMa表达下调，和上述预测一致；且RGMa下游因子FAK(Tyr397)磷酸化和Ras蛋白表达增加，与MFS及TLE有关，但具体机制仍不清楚。本项目拟运用基因转染、反义寡核苷酸技术等，结合新近发展的DREADD技术激活齿状回颗粒细胞，检测miR-20a-5p调控RGMa相关通路的主要分子及颗粒细胞电生理变化等，试从MFS的起源探讨颗粒细胞的激活与MFS及TLE的关系及对上述通路的影响，阐明miR-20a-5p调控RGMa的相关通路在MFS及TLE中的作用及机制，为TLE的治疗寻找新的靶点。

基于miR-20a-5p调控RGMa的相关通路在颞叶癫痫中的作用及机制研究设想，本项目运用基因转染、反义寡核苷酸技术等，结合新近发展的DREADD技术激活齿状回颗粒细胞，对miR-20a-5p调控RGMa的相关通路在颞叶癫痫中的作用及机制进行探讨的研究。首先培养原代神经元，建立稳定表达及稳定沉默miR-20a-5p的大鼠海马神经元，进而利用荧光素酶检测miR-20a-5p调控RGMa相关通路的主要分子及颗粒细胞电生理变化等，证实了RGMa是miR-20a-5p的靶基因。在此基础上，进一步通过基因转染上调RGMa及ASO技术下调miR-20a-5p，结合DREADD技术，从MFS的起源证实了基于miR-20a-5p调控RGMa的相关通路在TLE中的作用及机制。将含h3MDq受体基因的慢病毒立体定位注射至海马齿状回，建立PTZ癫痫动物模型，DREADD等技术进一步证实大鼠海马齿状回颗粒细胞的激活与MFS呈正相关。综上，项目结果为TLE的治疗寻找新的靶点奠定了坚实的工作基础。项目资助发表SCI论文2篇，另投稿2篇。培养研究生5名，其中博士生2名，硕士生3名。项目经费57万元，支出37.1538万元，各项支出基本与预算相符。剩余经费19.8462万元，将用于目前仍未支付本项目相关费用及后续研究。

内容获取失败，请点击重试