解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (6): 761-767.doi: 10.3969/j.issn.0529-1356.2014.06.006

• 组织学胚胎学发育生物学 • 上一篇    下一篇

肿瘤坏死因子受体相关因子6在大鼠失神经支配后胫前肌与比目鱼肌中的表达变化

孙华林 陈燕飞 仇嘉颖 王超 顾晓松 丁斐*   

  1. 江苏省神经再生重点实验室，江苏 南通 226001
  • 收稿日期:2014-04-10 修回日期:2014-05-23 出版日期:2012-12-06 发布日期:2014-12-06
  • 通讯作者: 丁斐 E-mail:dingfei@ntu.edu.cn
  • 基金资助:

    863 计划;973 计划;国家自然科学基金资助项目;江苏省高校基础研究计划

Changes of tumor necrosis factor receptor-associated factor 6 expression in denervated tibialis anterior muscle and soleus muscle of rats

SUN Hua-lin CHEN Yan-fei QIU Jia-ying WANG Chao GU Xiao-song DING Fei*   

  1. Jiangsu Key Laboratory of Neuroregeneration，Nantong University，Jiangsu Nantong 226001，China
  • Received:2014-04-10 Revised:2014-05-23 Online:2012-12-06 Published:2014-12-06
  • Contact: DING Fei E-mail:dingfei@ntu.edu.cn

摘要：

目的 通过蛋白质组学方法分析失神经支配后胫前肌与比目鱼肌的蛋白表达变化，探讨肿瘤坏死因子受体相关因子6（TRAF6）在失神经支配后胫前肌与比目鱼肌中的表达变化及其意义。 方法 制备大鼠坐骨神经离断模型，通过同位素相对标记与绝对定量 (iTRAQ)技术方法分析胫前肌与比目鱼肌在失神经支配后的蛋白表达变化，比较差异表达的蛋白在胫前肌与比目鱼肌中的差异，筛选出在胫前肌与比目鱼肌中的表达变化差异显著的关键蛋白，并通过免疫印迹法验证其表达，进一步通过体外方法验证关键蛋白的生物学作用。 结果 失神经支配后比目鱼肌萎缩速度和程度明显大于胫前肌（P<0.05，n=20），蛋白质组学研究发现有30个蛋白在胫前肌与比目鱼肌中表达变化差异较为明显，这些蛋白主要包括代谢相关蛋白、伴侣蛋白、收缩蛋白以及信号分子等，其中TRAF6在失神经支配后比目鱼肌中表达显著上升，蛋白免疫印迹法检测结果也证实了蛋白质组学的结果，TRAF6的靶基因肌肉环状指蛋白1（MuRF1）和肌肉萎缩盒F基因（MAFbx)在失神经支配后比目鱼肌中的表达也高于其在胫前肌中的表达（P<0.05，n=6）；为了进一步探讨TRAF6对肌萎缩的影响，在肌管中转染TRAF6 siRNA和对照siRNA，然后用地塞米松诱导C2C12肌管萎缩，发现TRAF6 siRNA转染组肌管的直径明显大于转染对照siRNA组（P<0.05，n=6）；TRAF6、MuRF1和MAFBx在TRAF6 siRNA转染组肌管中的表达显著低于转染对照siRNA组（P<0.05，n=6），结果提示TRAF6 siRNA有效抑制了靶基因TRAF6的表达，也同时抑制了其下游靶基因MuRF1和MAFBx的表达。 结论 TRAF6在失神经支配后比目鱼肌中的表达量显著高于其在胫前肌中的表达，抑制TRAF6的表达可以减轻地塞米松引起的肌管萎缩，由此推测失神经支配后比目鱼肌萎缩较胫前肌严重可能与失神经支配后比目鱼肌中TRAF6的升高更显著有关。 

关键词: 神经损伤, 肌萎缩, 肿瘤坏死因子相关因子6, 免疫印迹法, 大鼠

Abstract:

Objective To explore differentially expressed proteins between tibialis anterior muscle and soleus muscle after denervation by proteomics, and to analyze the changes of tumor necrosis factor receptor-associated factor 6（TRAF6）expression and its significance between tibialis anterior muscle and soleus muscle after denervation. Methods Rat models were prepared by cutting sciatic nerve. The protein expression changes between tibialis anterior muscle and soleus muscle after denervation were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) quantitative proteomics methods. The differentially expressed proteins were compared between tibialis anterior muscle and soleus muscle to screen key proteins regulating muscle atrophy. The expression was verified by Western blotting, and the biological role of key proteins was further validated in vitro. Results The atrophy of slow-twitch soleus muscle was faster than fast-twitch tibialis anterior muscle after denervation（P<0.05，n=20）. There were 30 proteins which displayed different expression between tibialis anterior muscle and soleus muscle. These differentially expressed proteins included metabolic-related proteins, chaperones, contractile proteins and signaling molecules. Among the including signaling molecules, TRAF6 displayed a significant increase in slow-twitch soleus muscle compared to fast-twitch tibialis anterior muscle after denervation, which was confirmed by Western blotting（P<0.05，n=6）. Muscle ring-finger protein-1（MuRF1）and muscle atrophy F-box（MAFbx), target genes of TRAF6, also showed a markly increase in slow-twitch soleus muscle compared to fast-twitch tibialis anterior muscle after denervation（P<0.05，n=6）. In order to further investigate the impact of TRAF6 in muscle atrophy, TRAF6 siRNA and control siRNA were tranfected into myotubes, which was then treated by dexamethasone. The results showed that the diameter of myotubes transfected with TRAF6 siRNA was significantly larger than that in the myotubes transfected with control siRNA（P<0.05，n=6）. The expression of TRAF6, MuRF1 and MAFBx in TRAF6 siRNA transfection group were significantly lower than that in the control siRNA transfected group（P<0.05，n=6）, suggesting that TRAF6 siRNA effectively inhibited the expression of the target gene TRAF6, also inhibited the expression of downstream its target genes of MuRF1 and MAFBx. Conclusion TRAF6 displays a significant increase in slow-twitch soleus muscle compared to fast-twitch tibialis anterior muscle after denervation. Inhibition of TRAF6 alleviates myotube atrophy induced by dexamethasone. Therefore, we speculate that the atrophy of slow-twitch muscle is faster than fast-twitch muscle after denervation, which is possible caused by the higher expression of TRAF6 in slow-twitch muscle after denervation.

Key words: Nerve injury, Muscle atrophy, Tumor necrosis factor receptor-associated factor 6, Western blotting, Rat