斑马鱼脊髓损伤后细胞因子和再生相关分子的表达分析

Expression analysis of cytokines and regeneration-related molecules after spinal cord injury in zebrafish

目的探讨成年斑马鱼脊髓损伤(spinal cord injury,SCI)后不同时间点细胞因子和再生相关分子的表达水平变化。方法56条斑马鱼随机分为脊髓损伤组(spinal cord injury group,SCI组,n=28)和假手术组(shamoperated group,Sham组,n=28),分别在SCI后的12 h、3 d、11 d和25 d进行组织取材(每个时间点n=7),脊髓组织的取材以损伤位点为中心,分为脊髓上段、中段(包含SCI位点)和下段,每段各2 mm。采用RT-qPCR法检测斑马鱼脊髓组织中肿瘤坏死因子(tumor necrosis factor-α,TNF-α)和白介素-10(interleukin-10,IL-10)和再生相关分子(thymosinβ4,TBETA4和annexin A2a,ANXA2A)的mRNA表达水平。结果相比于Sham组,损伤位点所在的脊髓中段在SCI后3 d,细胞因子TNF-α和IL-10表达水平显著降低,分别降低了93.5%和90%,再生相关分子TBETA4和ANXA2A的表达水平显著升高,分别升高了2.39倍和1.87倍;而在SCI后12 h和11 d/25 d,以上分子在脊髓中段的表达均无显著差异。提示SCI后3 d可能是损伤局部微环境改变的活跃期,因此,进一步检测和比较SCI后3 d的脊髓各段各基因的表达情况。相比于Sham组,细胞因子TNF-α和IL-10的表达水平仅在中段降低,上段及下段无显著差异;再生相关分子TBETA4的表达水平在上段升高了1.90倍,在下段升高了3.69倍;ANXA2A的表达水平在上段升高了3.45倍,在下段升高了2.63倍。结论斑马鱼SCI后3 d是损伤位点处细胞因子和再生相关分子发生显著变化的时间节点,在该时间点,细胞因子(TNF-α、IL-10)表达下调,其信号局限于损伤位点处,再生相关分子(ANXA2A、TBETA4)在脊髓的上段、中段和下段有不同程度的表达上调。

Objective To investigate the expression levels of cytokines and regeneration-related molecules after spinal cord injury(SCI) in adult zebrafish at different time points after injury.Methods 56 zebrafish were randomly divided into SCI group(n=28) and Sham group(n=28).Tissue was collected at 12 h,3 d,11 d and25 d after SCI(n=7 at each time point),and spinal cord tissue was divided into upper,middle(including injury site) and lower segments with 2 mm for each segment.RT-qPCR was used to detect mRNA expression levels of tumor necrosis factor-α(TNF-α),interleukin-10(IL-10) and regeneration-related molecules(thymosin beta 4,TBETA4 and annexin A2a,ANXA2A) in the spinal cord of zebrafish.Results At 3 d after SCI,the expression levels of cytokines TNF-α and IL-10 in the middle segment of the spinal cord was reduced by 93.5% and 90%respectively compared with the sham group,and the expression levels of regeneration-related molecule TBETA4 and ANXA2A was increased by 2.39 times and 1.87 times.While at 12 h and 11 d/25 d after SCI,the expression of the above molecules in the middle segment of spinal cord had no significant difference compared with the control group.This suggests that 3 days post-SCI may be the active period of local microenvironmental changes.Therefore,the expression of the above genes in 3 segments of spinal cord tissues at 3 days post-SCI was compared.It was found that the expression levels of inflammatory cytokines decreased in the middle segment and there is no significant difference in the upper and lower segments of the spinal cord compared with the Sham group.The expression levels of TBETA4 increased 1.90 and 3.69 times in the upper and lower segments,and the expression levels of ANXA2A increased 3.45 and 2.63 times in the upper and lower segments respectively at 3 d after SCI.Conclusion 3 days after SCI is the time point for significant changes of cytokines and regenerationrelated molecules at the injury site.At this time point,the signal of cytokines(TNF-α,IL-10) decreased and was limited to the injury site,while the signal of regeneration-related molecules(ANXA2A,TBETA4) was upregulated in upper,middle and lower spinal cord segment.