miR-429调控RhoE/PI3K通路诱导人成纤维细胞凋亡影响硬膜外瘢痕粘连的机制研究

Epidural scar adhesion after laminectomy is one of the important factors affecting the clinical curative effect. We found that the tacrolimus-induced apoptosis in fibroblast can prevent epidural scar adhesion. Gene chip technology showed that miR-429 is in low expression in fibroblasts after stimulated of tacrolimus. Previous studies have suggested that inhibiting the function of miR-429 can increase the rate of drug-induced apoptosis of fibroblast, bioinformatics analysis and luciferase reporter gene system verification results found and confirmed RhoE is the target genes of miR-429. RhoE is an important inhibitory factor of PI3K signaling pathway, which can induce apoptosis by inhibiting the cell cycle. This study picks miR-429 molecules as the breakthrough point, and it will use Western-Blot, qRT-PCR, Lentiviral Infection, animal models and so on to clarify the exact molecular mechanisms of miR-429-induced fibroblast apoptosis by RhoE/PI3K pathway. This study helps to clear miR-429 in the role of fibroblast apoptosis and the molecular mechanisms of the prevention of epidural scar adhesion. Also, it provides new ideas and potential therapeutic target to prevent and cure epidural scar adhesion.

椎板切除术后硬膜外瘢痕粘连是影响临床疗效的重要原因之一。我们发现他克莫司能通过诱导成纤维细胞凋亡预防硬膜外瘢痕粘连，基因芯片结果显示miR-429低表达于他克莫司刺激后的人成纤维细胞。前期研究发现抑制miR-429功能可以增加药物诱导成纤维细胞的凋亡率，生物信息学分析及荧光素酶报告基因系统验证结果发现并证实RhoE是miR-429的靶基因。RhoE是PI3K信号通道的重要抑制因子，可通过抑制细胞周期诱导细胞凋亡。本课题拟以miR-429为切入点，采用Western-Blot、qRT-PCR、慢病毒转染、动物体内实验等方法，从体内、体外研究阐述miR-429 调控RhoE/PI3K通道诱导成纤维细胞凋亡减少硬膜外瘢痕粘连的确切机制。本研究有助于明确miR-429在人成纤维细胞凋亡中的作用及其预防硬膜外瘢痕粘连的分子学机制，为硬膜外瘢痕粘连的防治防治提供新思路及潜在治疗靶点。

研究背景和目的：成纤维细胞过度增殖是硬膜外瘢痕粘连形成的主要原因。既往研究表明他克莫司（FK506）能诱导成纤维细胞凋亡预防硬膜外瘢痕粘连。本研究旨在探讨miR-429在成纤维细胞中的生物学特性及其影响硬膜外瘢痕粘连的机制。.实验方法：CCK-8检测FK506对人成纤维细胞活性的影响。TUNEL，AV/PI和Western-blot检测FK506诱导人成纤维细胞凋亡的情况。RT-qPCR检测FK506和TGF-β1处理人成纤维细胞后miR-429的表达情况。荧光素酶报告验证RhoE和miR-429的靶向对应关系。CCK-8，EdU和AV/PI检测过表达和沉默miR-429、沉默RhoE对人成纤维细胞增殖、凋亡的影响及可能的机制；组织学和TUNEL染色评价miR-429与硬膜外瘢痕粘连的关系。.实验结果：CCK-8结果显示FK506显著降低人成纤维细胞的活性，呈现出浓度和时间依赖性。TUNEL，AV/PI和Western-blot结果显示FK506能诱导人成纤维细胞发生凋亡，且AKT通路参与其中。RT-qPCR结果显示FK506能下调miR-429表达，而TGF-β1能上调miR-429表达。EdU，AV/PI和Western-blot结果显示下调miR-429表达能抑制人成纤维细胞的增殖，诱导人成纤维细胞的凋亡，也发现AKT通路参与其中。荧光素酶报告提示RhoE是miR-429的靶基因，慢病毒敲减RhoE表达后能减少FK506对人成纤维细胞凋亡的作用，并且也发现AKT通路参与其中。动物实验结果显示下调miR-429表达（FK506和miR-429 inhibitor）能诱导成纤维细胞凋亡，预防大鼠硬膜外瘢痕粘连的形成。.结论：下调miR-429表达，上调靶基因RhoE表达能抑制人成纤维细胞增殖，诱导人成纤维细胞凋亡，预防大鼠硬膜外瘢痕粘连，且AKT通路参与其中。本研究结果可为硬膜外瘢痕粘连的防治提供新思路和潜在治疗靶点。