坐骨神经预损伤后背根神经节中miRNomes改变对大鼠脊髓后索损伤修复的影响

Effect of alteration of miRNAomes in rat dorsal root ganglia after sciatic nerve conditioning injury on the repairment of dorsal column lesion

中文关键词:  【关键词】 坐骨神经预损伤  脊髓后索损伤  miRNA  Dusp4  p38蛋白  大鼠

英文关键词:【Key words】 Sciatic nerve conditioning injury  Dorsal column lesion  miRNA  Dusp4  p38 protein  Rat

中文摘要:

  【摘要】 目的：研究大鼠坐骨神经预损伤后背根神经节中miRNomes改变对脊髓后索损伤修复的影响。方法：39只雌性Wistar大鼠随机分为A、B、C、D组。A组（n=12）坐骨神经损伤造模后7d进行T10节段脊髓后索损伤造模，B组（n=12）仅进行T10节段脊髓后索损伤造模，C组（n=12）仅进行坐骨神经损伤造模，D组（n=3）不进行任何造模操作。A组和B组分别于脊髓后索损伤造模后4h、3d、7d、14d取背根神经节行总RNA提取和Western blot检测，于脊髓后索损伤造模后14d取损伤中心脊髓组织行神经丝蛋白200（NF-200）免疫组织化学染色和HE染色；C组于A组各时间点取材的同时取背根神经节行总RNA提取和Western blot检测；D组取背根神经节行总RNA提取和Western blot检测。对A、B两组各时间点背根神经节miRNA表达谱进行微阵列芯片分析和生物信息学分析，观察与坐骨神经预损伤促进脊髓后索损伤修复有关的miRNA，选出A组中与B组相比变化倍数明显、经过生物信息学分析靶蛋白为Dusp4的miR-199a-5p进行研究。并用RT-qPCR技术对各组miR-199a-5p及A、B和D组Dusp4 mRNA表达进行检测，用Western blot技术检测各组Dusp4蛋白以及A、D组p38蛋白和p-p38蛋白，对A组和B组脊髓后索损伤中心脊髓组织用NF-200免疫组织化学染色及HE染色观察损伤脊髓的恢复情况。结果：芯片分析结果显示miR-199a-5p在A组各个时间点表达与D组相比明显下调，B组miR-199a-5p在脊髓后索损伤后4h表达与D组相比上调，3d、7d和14d的表达量无明显变化。RT-qPCR结果显示A组各时间点miR-199a-5p表达与D组相比下调（P＜0.05），B组miR-199a-5p在脊髓后索损伤后4h表达与D组相比上调（P＜0.05），3d、7d和14d的表达量与D组比较无明显变化，C组各时间点miR-199a-5p表达与D组比较无明显变化。A组和B组各时间点Dusp4 mRNA表达与D组相比无明显变化。A组Dusp4蛋白在脊髓后索损伤后各个时间点与D组比较均有上调且存在统计学差异（P＜0.05）。B组Dusp4蛋白在脊髓后索损伤后4h表达与D组相比显著下调（P＜0.05），脊髓后索损伤后3d、7d、14d与D组比较无明显差异。C组Dusp4蛋白在各时间点的表达水平与D组比较无明显差异。A组p38蛋白及p-p38蛋白的表达变化趋势与miR-199a-5p趋势一致。在脊髓后索损伤后14d，与B组比较A组损伤中心尾端脊髓NF-200表达明显增加，且损伤中心尾端脊髓白质纤维束形态规整、后索纤维束排列有序。结论：大鼠坐骨神经预损伤后背根神经节中miR-199a-5p表达下调可以促进脊髓后索损伤的修复。

英文摘要:

  【Abstract】 Objectives: To study the effect of alteration of miRNomes after sciatic nerve conditioning injury on the repairment of dorsal column lesion. Methods: Thirty-nine female Wistar rats were divided randomly into four groups, group A, B, C and D. Group A(n=12) underwent dorsal column lesion on the 10th thoracic vertebra at the 7th day after sciatic nerve conditioning injury. Group B(n=12) underwent only dorsal column lesion. Group C(n=12) underwent only sciatic nerve injury. Group D(n=3) was used as blank control. The dorsal root ganglias of group A and B were excised for total RNA isolation and western blot at 4 hours, 3 days, 7 days and 14 days after dorsal column lesion. The spinal cord tissues of lesion sited in group A and B were harvested at 14 days after dorsal column lesion for immunohistochemistry of neurofilament-200(NF-200) and hematoxylin-eosin staining. The dorsal root ganglias of group C were harvested at same time points for total RNA isolation and Western blot. The dorsal root ganglias of group D were harvested for total RNA isolation and Western blot. To study its mechanism, the miRNA profiles of dorsal root ganglias in group A and B were investigated by Microarray and bioinformatics. The significantly changed miRNA miR-199a-5p whose target was Dusp4 was screened out. RT-qPCR was used to detect the expression of miR-199a-5p in each group and the expression of Dusp4 mRNA in group A, B and D. Western blot was applied to test the expression of Dusp4 protein in each group and the expression of p38 protein and p-p38 protein in group A and D. Immunohistochemistry of NF-200 and hematoxylin-eosin staining were used to investigate the repairment of dorsal column lesion. Results: Microarry revealed that miR-199a-5p downregulated at each time point of group A and upregulated at 4h of group B, however the expression of miR-199a-5p in group B did not alter significantly at 3d, 7d, and 14d after dorsal column lesion compared with that of group D. RT-qPCR showed that miR-199a-5p downregulated at each time point of group A(P＜0.05) and upregulated at 4h of group B(P＜0.05), while the expression of miR-199a-5p at 3d, 7d and 14d of group B and the expression at each time point of group C showed no statistical difference compared with that of group D. The Dusp4 mRNA expressions of group A and B showed no statistical difference at each time point compared with that of group D. Statistical difference was noted for Dusp4 protein at each time point of group A with group D(P＜0.05). Compared with group D, the Dusp4 protein downregulated significantly at 4h of group B(P＜0.05) and then raised to the level of group D at 3d, 7d and 14d. Compared with group D, there was no statistical difference regarding to the expression of Dusp4 protein at each time point of group C. p38 protein and phosphorylated p38 protein level was consistent with the level of miR-199a-5p in group A. At 14 days, compared with group B, the expression level of neurofilament protein increased significantly and the shape of nerve fiber bundle was more regular in caudal lesion site of group A. Conclusions: The downregulation of the expression of miR-199a-5p after sciatic nerve conditioning injury can promote the repairment of dorsal column lesion.