RNA-Seq筛选脊髓损伤后的差异基因及部分基因功能分析

发布时间：2018-03-26 13:08

本文选题：脊髓损伤　切入点：血红素氧化酶　出处：《解剖学报》2017年02期

【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 to analyze the difference of gene expression in different stages of spinal cord injury by RNA-Seq. Methods Forty-eight Spinal Cord injury (sci) rats were randomly divided into two groups: sham operation group and experimental group. The RNA-Seq technique was used to screen the differentially expressed genes at different time points after spinal cord injury, and the expression pattern, functional annotation and pathway analysis of the differentially expressed genes were analyzed. Heme oxygenase 1 (Hmox1) and other interested genes were screened for mRNA and protein level verification and functional analysis. Results RNA-Seq results showed that, compared with sham operation group, The number of genes significantly changed at 4 and 7 days after spinal cord injury was 944, 1362 and 1421, respectively. The changes of these genes at different time points after spinal cord injury could be roughly divided into 8 forms. Real-time PCR results showed that Hmox1, urokinase plasminogen excimer and urokinase plasminogen exciter. The expression changes of Plaun, plasminogen activator inhibitor Serpine 1 and neutrophil cytoplasmic factor ncf2) were similar to those of RNA-Seq sequencing. Western blotting analysis showed that the expression of Plaun, plasminogen activator inhibitor, and neutrophil cytoplasmic factor (Ncf2) were similar to those of RNA-Seq sequencing. The change trend of Hmox1 protein expression was consistent with the nucleic acid level after spinal cord injury, the immunohistochemical results showed that Hmox1 was mainly expressed in neurons of spinal cord tissue after injury. Conclusion RNA-Seq technique can efficiently analyze the differential genes after spinal cord injury. The identification and functional analysis of genes of interest selected from a large number of differentially expressed genes provide some information for elucidating the molecular mechanism of spinal cord injury.
【作者单位】：南通大学医学院组织学与胚胎学系;南通大学附属医院生殖医学中心;济宁市第一人民医院生殖医学科;江南大学无锡医学院基础医学院;
【基金】：国家自然科学基金(81271721,81200828,81501610) 江苏高校优势学科建设工程资助项目
【分类号】：R651.2

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