解剖学报

解剖学报 ›› 2018, Vol. 49 ›› Issue (1): 14-19.doi: 10.16098/j.issn.0529-1356.2018.01.003

• 神经生物学 • 上一篇    下一篇

皮质脊髓束损伤后胶质细胞激活相关microRNA的筛选与验证

刘晓东1,2 王艺儒1 吕金阳1 徐晓燕1 张致祎1 王鑫1 吕广明1,3*   

  1. 1.南通大学医学院解剖学教研室; 2.南通市第6人民医院医务科;  3.南通大学神经再生重点实验室，江苏 南通 226001
  • 收稿日期:2017-03-06 修回日期:2017-05-31 出版日期:2017-02-06 发布日期:2018-02-06
  • 通讯作者: 吕广明 E-mail:915984810@qq.com
  • 基金资助:
    国家自然科学基金重点项目;国家级大学生创新创业训练计划项目

Screening and identification of the glial cell activation associated microRNA after corticospinal tract injury

LIU Xiao-dong 1,2 WANG Yi-ru1 Lü Jin-yang1 XU Xiao-yan1 ZHANG Zhi-yi1 WANG Xin1 Lü Guang-ming 1,3*   

  1. 1.Department of Human Anatomy, Medical School of Nantong University; 2.Medical Department, the Sixth People’s Hospital of Nantong; 3.Jiangsu Key Laboratory of Neuroregeneration, Jiangsu Nantong 226001, China
  • Received:2017-03-06 Revised:2017-05-31 Online:2017-02-06 Published:2018-02-06
  • Contact: Lü Guang-ming E-mail:915984810@qq.com

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摘要：

目的 检测皮质脊髓束损伤头侧和尾侧microRNA及其靶基因mRNA的表达变化，探讨microRNA在皮质脊髓束损伤修复过程中的生物学作用。 方法 在左侧延髓锥体切断大鼠锥体束（30只），建立单侧大鼠皮质脊髓束损伤模型，采用BBB评分进行行为学评价；实验以左侧大鼠皮质脊髓束损伤模型作为研究对象，利用生物信息学方法筛选差异表达的microRNA预测靶基因与mRNA的差异筛选取交集，进行microRNA和mRNA共表达分析；Real-timePCR检测12只大鼠miR-342-5p、Irf8的表达水平；Western blotting检测6只大鼠Irf8、Iba1蛋白的表达变化。 结果 皮质脊髓束损伤后观察到右后肢运动不协调，BBB评分表明运动功能受限，但是6只大鼠没有完全瘫痪，损伤后2 h, 1、3、5和7 d BBB评分与对照组相比差异有统计学意义（P<0.05），提示模型制备成功。头侧和尾侧5 d与2h 相比，聚集于与修复再生相关的生物学过程。Real-time PCR结果显示，尾侧5 d与2h相比，miR-342-5p表达差异有显著性（P<0.01），并且与Irf8呈现反向调节关系，而在头侧miR-342-5p表达差异无统计学意义（P>0.05）。Real-time PCR数据分析显示，Irf8与芯片结果一致，与对照组相比差异有统计学意义(P<0.01)。Western blotting 结果显示，5 d和2h相比尾侧Irf8的表达显著增加，Iba1的表达明显增高，差异有统计学意义（P<0.01）。 结论 皮质脊髓束损伤后损伤头侧和尾侧均有显著性上调或下调的差异表达基因，在损伤尾侧miR-342-5p和Irf8呈反向调节，提示miR-342-5p与胶质细胞激活从而促进脊髓损伤修复再生密切相关。

关键词: 皮质脊髓束, 再生, microRNA, 差异表达基因, 基因-基因网络, 免疫印迹法, 大鼠

Abstract:

Objective To detect changes in microRNA and target mRNA at the head and tail of the corticospinal tract (CST) injury side, and to investigate the function of microRNA in spinal cord injury (SCI) and repairment, which could provide a theoretical basis and experimental evidence in regeneration of the CST after injury.Methods The left side of the CST in 30 rats was cut at the level of the pyramid of medullar oblongata. The BBB test was used to assess the motor function. After the CST injury, the bioinformatics method was conducted to screen differences in microRNA target gene prediction. The intersection of differentially expressed mRNA was selected to conduct the microRNA and mRNA co-expression analysis. Real-time PCR was applied to detect the gene expression of miR-342-5p and Irf8 in 12 rats. Western blotting was applied to detect the protein expression of Irf8 and Iba1 in 6 rats. Results The limb movement after CST semitransection was uncoordinated, and motor function was limited according to BBB test but not completely paralyzed. There were statistical significances(P<0.05)after comparing 2 hours, 1 day, 3 days, 5 days and 7 days with the control group, indicating the successful establishment of model. Comparison of the inrostraland caudal region between 2 hours and 5 days, we focused on biological processes in regeneration. Data analysis from Realtime PCR experiments indicated that caudal miR-342-5p expression was significantly different(P<0.01) from 2 hours to 5 days, with a reverse correlation with Irf8 expression, whereas no change was observed in rostral miR-342-5p(P>0.05). Real-time PCR data analysis showed that the expression of Irf8 was consistent with the chip result , statistically significant difference compared with control groups (P<0.01). The result of Western blotting indicated that, comparing tissue from 5 days with 2 hours, both Irf8 and Iba1 were significantly creased in caudal samples (P<0.01). Conclusion After CST injury, there were significant up-regulated or down-regulated differentially expressed genes in rostral and caudal regions. In the caudal region, the miR-342-5p and Irf8 reverse expression and miR-342-5p was closely related to SCI repair and regeneration by glial cell activation.

Key words: Corticospinal tract, Regeneration, Micro RNA, Differentially expressed gene, Gene-gene network, Western blotting, Rat

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