RNA-Seq筛选脊髓损伤后的差异基因及部分基因功能分析

陈颖 王迪 牛学英王晓冬 陈雪*

doi:10.16098/j.issn.0529-1356.2017.02.002

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解剖学报 ›› 2017, Vol. 48 ›› Issue (2) : 128-134. DOI: 10.16098/j.issn.0529-1356.2017.02.002

神经生物学

RNA-Seq筛选脊髓损伤后的差异基因及部分基因功能分析

陈颖¹王迪³ 牛学英^1,4王晓冬¹陈雪^1,2*

作者信息 +

Screening the differentially expressed genes after spinal cord injury by RNA-Seq and subsequent functional analysis for partial candidate genes

CHEN Ying¹WANG Di³NIU Xue-ying ^1,4 WANG Xiao-dong¹CHEN Xue ^1,2*

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文章历史 +

摘要

目的应用RNA-Seq技术分析脊髓损伤后不同阶段基因表达差异情况。方法使用IH脊髓打击仪制备脊髓损伤模型，SD大鼠48只随机分成两组，假手术组和实验组，实验组分别于损伤后1d、4d和7d取材。运用RNA-Seq技术筛选脊髓损伤后不同时间点的差异表达基因，对差异基因进行表达模式分析，功能注释，通路分析等，筛选出血红素氧化酶1（Hmox1）等感兴趣基因进行mRNA和蛋白水平的验证及功能分析。结果 RNA-Seq结果表明，与假手术组相比，脊髓损伤后1d、4d和7d发生显著变化的基因分别有944、1362和1421个。上述差异基因在损伤后不同时间点的变化趋势大致可分为8种形式。Real-time PCR结果显示，Hmox1、尿激酶型纤溶酶原激活剂（Plau）、纤溶酶原激活物抑制剂（Serpine1）和中性粒细胞胞质因子（Ncf2）的表达变化与RNA-Seq测序结果基本一致。Western blotting结果显示，脊髓损伤后Hmox1的蛋白表达变化趋势与核酸水平一致；免疫组织化学结果显示，损伤之后Hmox1主要表达于脊髓组织的神经元中。结论 RNA-Seq技术能高效分析脊髓损伤后的差异基因，从大量差异基因中筛选出感兴趣基因的验证和功能分析为阐述脊髓损伤的分子机制提供了部分资料。

Abstract

Objective We apply RNA-Seq technology to characterize the temporal changes in global gene expression after spinal cord injury (SCI) in rats. Methods Spinal cord contusion injury was produced with the Infinite Horizon Device. A total of 48 rats were randomly divided into sham control, and contusion injury for 1 day, 4 days and 7days. RNA-Seq technology was carried out to screen the differentially expressed genes (DE genes) after SCI. We also performed expression pattern and pathway analysis for the DE genes, and selected the candidates to further expression variation validation. Results Compared with sham group, there were 944DE genes at the first day, 1362 DE genes at the 4th day and 1421 DE genes at the 7th day. The expression variation patterns were roughly divided into 8 kinds of forms.In addition, Real-time PCR results showed that the expression patterns of heme-oxygenases 1（Hmox1）, Plau, Serpine1 and Ncf2 were consistent with RNA-seq analysis. The result of immunohistochemistry showed that Hmox1 was highly expressed in spinal cord neurons after injury. Conclusion RNA-Seq analysis is useful to screen the DE genes after SCI, and the validated genes could partially explain the molecular mechanism of SCI.

导出引用

陈颖王迪牛学英王晓冬陈雪. RNA-Seq筛选脊髓损伤后的差异基因及部分基因功能分析[J]. 解剖学报. 2017, 48(2): 128-134 https://doi.org/10.16098/j.issn.0529-1356.2017.02.002

CHEN Ying WANG Di NIU Xue-ying WANG Xiao-dong CHEN Xue. Screening the differentially expressed genes after spinal cord injury by RNA-Seq and subsequent functional analysis for partial candidate genes[J]. Acta Anatomica Sinica. 2017, 48(2): 128-134 https://doi.org/10.16098/j.issn.0529-1356.2017.02.002

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