微RNA-21调控脊髓损伤后成纤维细胞功能的研究

MicroRNA-21-5p promotes fibrosis of spinal fibroblasts after spinal cord injury

目的明确脊髓损伤后微RNA(microRNA,miR)-21对脊髓成纤维细胞的调控作用,并探索miR-21在脊髓损伤病理过程中发挥作用的机制。方法采用免疫荧光法鉴定脊髓成纤维细胞,划痕法建立细胞损伤模型,实时荧光定量逆转录聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)测定损伤后脊髓成纤维细胞中miR-21以及纤维化相关基因的表达变化。过表达和抑制小鼠脊髓成纤维细胞中的miR-21,qRT-PCR和蛋白质印迹分析法(Western Blot,WB)测定纤维化和凋亡、增殖相关指标的表达水平。结果脊髓成纤维细胞划痕损伤后miR-21的表达升高(P<0.05),成纤维细胞活化相关基因表达升高(P<0.05),反之亦然;成纤维细胞的增殖指标随miR-21的表达一致,成纤维细胞的凋亡指标与miR-21的表达情况相反。结论miR-21对机械损伤后脊髓成纤维细胞纤维化有增强作用,同时可以增强脊髓成纤维细胞的增殖并抑制其凋亡。miR-21与脊髓损伤后纤维瘢痕形成关系密切,这也为脊髓损伤后功能恢复提供了一个潜在的治疗靶点。

Objective To investigate the regulatory effect of miRNA-21-5 p(miR-21)on spinal fibroblasts,and to explore the mechanism of miR-21 related pathological process of spinal cord injury.Methods Spinal cord fibroblasts were identified by immunofluorescence.Spinal fibroblasts damage model was established by scratch method.Quantitative real-time polymerase chain reaction(RT-PCR)was used to determine the relative expression of miR-21 and fibrosisrelated genes in spinal cord fibroblasts after injury.The expression of miR-21 in spinal cord fibroblasts was up-regulated and down-regulated by using miR-21 mimics/inhibitor,and the expression levels of apoptosis and proliferation-related proteins were detected by Western Blot(WB).Results The expression of miR-21 and fibrosis-related genes were increased after spinal cord fibroblast scratch(P<0.05).Up-regulation of the miR-21 can increase the expression of apoptosis-related genes in fibroblasts(P<0.05),and vice versa.The proliferation of fibroblasts was consistent with the expression of miR-21,while the apoptosis of fibroblasts was contrary to the expression of miR-21.Conclusions miR-21 enhanced the fibrosis and proliferation,inhibited the apoptosis of spinal cord fibroblasts after mechanical injury.This indicates that miR-21 is closely related with the formation of fibrotic scar after spinal cord injury,which also provides a potential therapeutic target for spinal cord injury.