中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (9): 1583-1593.doi: 10.4103/1673-5374.255994

• 原著:脊髓损伤修复保护与再生 • 上一篇    下一篇

第2代测序技术RNA-Seq筛选脊髓损伤后差异表达的基因

  

  • 出版日期:2019-09-15 发布日期:2019-09-15
  • 基金资助:

    中国国家自然科学基金项目（31570983），江苏省高等学校优先学科项目

RNA sequencing screening of differentially expressed genes after spinal cord injury

Yi Li 1, 2 , Ying Chen 2 , Xiang Li 2 , Jian Wu 2 , Jing-Ying Pan 2 , Ri-Xin Cai 2 , Ri-Yun Yang 2 , Xiao-Dong Wang 2, 3   

  1. 1 School of Biology & Basic Medical Sciences, Soochow University, Suzhou, Jiangsu Province, China
    2 Department of Histology and Embryology, Medical College, Nantong University, Nantong, Jiangsu Province, China
    3 Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China
  • Online:2019-09-15 Published:2019-09-15
  • Contact: Xiao-Dong Wang, MD, wxdzw@ntu.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 31570983 (to XDW); the Priority Academic Program Development of Jiangsu Higher Education Institutes of China.

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摘要：

在探索脊髓损伤的治疗方法时，有必要了解脊髓损伤涉及的关键基因及其相应的调节网络；然而既往研究使用盲法选择和分析一两个基因不能完整揭示脊髓损伤的复杂分子生物学机制，作为第2代测序技术的RNA-Seq技术可以更好地进行全基因组测序。(1)实验对打击T8脊髓建立的脊髓损伤大鼠模型的损伤脊髓组织进行全基因组测序分析，显示脊髓损伤后第1，4和7天发生显著变化的基因数分别为944，1362和1421。(2)在基因本体论分析和差异表达基因的时间表达分析后，以实时PCR分析和Western blot分析对C5ar1，Socs3和Ccl6基因的mRNA和蛋白变化进行验证。其结果证实C5ar1，Socs3和Ccl6 mRNA与RNA-Seq测序结果一致，C5ar1蛋白表达趋势与基因也是一致的，且C5ar1主要在神经元和星形胶质细胞中表达。(3)通过Chilibot数据库和文献检索发现可能Itgb2与C5ar1可能有关，免疫荧光染色显示Itgb2的表达与C5ar1高度一致，且Itgb2主要分布于星形胶质细胞中。(4) RNA-Seq技术可以相对全面地筛选脊髓损伤后不同时间点的差异表达基因，经过分析验证可以从中筛选出与脊髓损伤密切相关基因，这可以为进一步阐述脊髓损伤的分子机制提供实验数据，还能为脊髓损伤治疗提供可能的靶点。实验于2018-03-06经江苏省实验动物伦理委员会批准。

orcid: 0000-0003-4813-6858 (Xiao-Dong Wang)

关键词: 脊髓损伤, RNA-Seq技术, C5ar1, Itgb2, Socs3, Ccl6, 星形胶质细胞, 神经再生

Abstract:

In the search for a therapeutic schedule for spinal cord injury, it is necessary to understand key genes and their corresponding regulatory networks involved in the spinal cord injury process. However, ad hoc selection and analysis of one or two genes cannot fully reveal the complex molecular biological mechanisms of spinal cord injury. The emergence of second-generation sequencing technology (RNA se¬quencing) has provided a better method. In this study, RNA sequencing technology was used to analyze differentially expressed genes at different time points after spinal cord injury in rat models established by contusion of the eighth thoracic segment. The numbers of genes that changed significantly were 944, 1362 and 1421 at 1, 4 and 7 days after spinal cord injury respectively. After gene ontology analysis and temporal expression analysis of the differentially expressed genes, C5ar1, Socs3 and CCL6 genes were then selected and identified by real-time polymerase chain reaction and western blot assay. The mRNA expression trends of C5ar1, Socs3 and CCL6 genes were consis¬tent with the RNA sequencing results. Further verification and analysis of C5ar1 indicate that the level of protein expression of C5ar1 was consistent with its nucleic acid level after spinal cord injury. C5ar1 was mainly expressed in neurons and astrocytes. Finally, the gene Itgb2, which may be related to C5ar1, was found by Chilibot database and literature search. Immunofluorescence histochemical results showed that the expression of Itgb2 was highly consistent with that of C5ar1. Itgb2 was expressed in astrocytes. RNA sequencing technology can screen differentially expressed genes at different time points after spinal cord injury. Through analysis and verification, genes strongly as¬sociated with spinal cord injury can be screened. This can provide experimental data for further determining the molecular mechanism of spinal cord injury, and also provide possible targets for the treatment of spinal cord injury. This study was approved ethically by the Labo¬ratory Animal Ethics Committee of Jiangsu Province, China (approval No. 2018-0306-001) on March 6, 2018.

Key words: nerve regeneration, spinal cord injury, RNA sequencing, C5ar1, Itgb2, Socs3, CCL6, astrocytes, rats, neural regeneration