miR-21-5p靶向调控EPO-R通路在脊髓损伤后胶质瘢痕形成中的作用机制研究

The formation of glial scar has an effect of mechanical barrier and biological inhibition in the growth and recanalization of nerve fibers during the subacute and chronic period of spinal cord injury (SCI).It has been confirmed that the reactive astrocytes (RAS) is the cellular basis in the formation of glial scar.Also miR-21-5p,as one of the important regulatory factors in RAS,can reduce the size of RAS and decrease the expression of GFAP,which has a reactivity increasing in a time dependent after SCI. So the study in signaling pathway of miR-21-5p on effects of RAS has a vital improtant role in effective controling the formation of glial scar during the subacute stage. Our previous study confirmed that EPO-R plays a positive role in SCI. On this basis, we put forward a hypothesis that miR-21-5p has an effect on RAS through regulation of EPO-R signaling pathway in the subacute stage of SCI.Our research, with the application of GFAP-Cre/ROSA-GFP transgenic mice, focuses on the study of miR-21-5p regulating EPO-R signaling pathway through in vivo and in vitro, especially the time periodic law on the effect of RAS at the subacute phase after SCI,making clear of the functional targets in the downstream.The desired results will reveal the time window conversed from playing a positive to a negative role respectively in the acute and subacute stage of SCI ,and also the mechanism in the formation of glial scar on molecular level, and eventually to provide theoretical basis for clinical intervention and further enrich the theory of regulation between RAS and miR-21-5p.

在脊髓损伤（SCI）亚急性期后胶质瘢痕形成对神经纤维生长及再通起机械屏障和生物抑制作用。已证实反应性星形胶质细胞（RAS）是胶质瘢痕形成的细胞基础，而miR-21-5p是RAS的重要调控因素之一，其在SCI后呈时间依赖性反应性增多，可以减小RAS尺寸，降低GFAP表达。因此探讨miR-21-5p在这一时段对RAS作用的信号通路，对于有效控制亚急性期后胶质瘢痕形成至关重要。本研究在前期证实EPO-R在SCI中发挥作用的基础上，提出SCI亚急性期后miR-21-5p通过调控EPO-R信号通路而作用于RAS的假说。本课题重点研究SCI亚急性期后miR-21-5p调控EPO-R信号通路对RAS作用的时间阶段性规律，明确下游发挥作用的功能性靶标。预期结果的取得将从分子水平揭示胶质瘢痕形成在SCI急性和亚急性期分别发挥积极和不利作用的转换时间窗及作用机制，为临床干预治疗提供理论依据。

在脊髓损伤（SCI）亚急性期后胶质瘢痕形成对神经纤维生长及再通起机械屏障和生物抑制作用。已证实反应性星形胶质细胞（RAS）是胶质瘢痕形成的细胞基础，而miR-21-5p是RAS的重要调控因素之一，其在SCI后呈时间依赖性反应性增多，可以减小RAS尺寸，降低GFAP表达。本课题组在体外用C57来源的星形胶质细胞通过TGF-β刺激法构建脊髓损伤胶质瘢痕体外模型，之后用siRNA干预和miR-21过表达/抑制的慢病毒载体验证miRNA-21对细胞模型的影响，并探索了miR-21调节脊髓损伤后胶质瘢痕形成的作用通路，在体内手术处理C57BL/6小鼠构建脊髓损伤瘢痕模型，随机分为正常组、假手术组和实验组，实验组用miRNA-21过表达和抑制慢病毒分别处理，在体内验证miRNA-21的影响及作用通路。实验证明，脊髓损伤后miRNA-21和TGF-β1的表达水平明显增加，GFAP和CSPG等星形胶质细胞标记物表达水平明显下降。同时敲低miRNA-21的表达水平可以减少GFAP和CSPG的表达。其中，我们发现PTEN是miRNA-21的靶基因之一，miR-21-5p可能通过调节PTEN的表达来调节脊髓损伤后星形胶质细胞的增殖和凋亡。总之，microRNA-21-5p靶向PTEN通过PI3K/AKT/mTOR通路调节脊髓损伤后的胶质瘢痕形成，这对于开发新的改善脊髓损伤后功能恢复的治疗方法具有启示性意义。