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应用cDNA微阵列技术筛选大鼠脊髓损伤修复相关基因

肖林, 马振莲, 李欣, 林秋霞, 阙海萍, 刘少君

军事医学科学院基础医学研究所神经生物学研究室.北京 100850

摘要

脊髓损伤是一类常见的、高致残率的中枢神经系统疾病,由于多种复杂因素影响其损伤后的修复过程,损伤脊髓的再生能力非常有限。研究采用cDNA微阵列技术筛选大鼠脊髓损伤后出现的差异表达基因。实验组动物在T8--T9进行脊髓全横断手术，对照组动物只打开椎板；4.5 d后取脊髓进行RNA提取并在反转录过程中进行Cy3/Cy5标记，然后与预制的、带有4 041条特异性探针的芯片进行杂交。Cy5/Cy3信号比值≥2.0视为脊髓损伤后出现差异表达的基因。通过筛选，作者得到了65个上调表达基因(21个已知基因,30个已知EST和14个未知基因)和79个下调基因(20个已知基因，42个已知EST和17个未知基因)。进一步通过半定量RT--PCR对其中的5个上调已知基因(Timp1,Tagln,Vim,Fc gamma receptor.Ctss)和三个下调已知基因(stearyl-CoA desaturase,F2,Ensa)的表达情况进行了验证，结果显示与芯片结果一致。这些基因可能在脊髓损伤后的修复过程中起一定的作用，对其深入研究将有助于揭示脊髓损伤修复的分子机制。

关键词： 脊髓; 基因表达; cDNA微阵列; 反转录PCR

cDNA microarray analysis of spinal cord injury and regeneration related genes in rat

Xiao Lin, Ma Zhenlian, Li Xin, Lin Qiuxia, Que Haiping, Liu Shaojun

Department of Neurobiology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences.Beijing 100850

Abstract

The acute traumatic spinal cord injury (SCI) is a commonly seen and severe case in clinic. However, the repair and regeneration of injured spinal cord is limited. This is likely due to that different kinds of factors are involved in regeneration after SCI.In the present study, we used complementary DNA microarray consisting of 4 041 specific probes from rat to identify genes that were differentially expressed after SCI. The animals were subjected to complete transection injury of the thoracic spinal cord (T8-T9). Sham operated animals Receivedonly a laminectomy. Four and a half days later, rat spinal cord was dissected out for total RNA isolation. The fluorescent (Cy3 and Cy5) labeled probes were prepared and hybridized to the microarray. Genes that showed 2-fold difference in SCI tissue were identified. Sixty-five up-regulated genes consisted of 21 known genes, 30 known expressed sequence tags (ESTs) and 14unknown genes. Seventy-nine down-regulated genes comprised 20 known genes, 42 known ESTs and 17 unknown genes. In 41differentially expressed known genes, 5 up-regulated genes, i.e., tissue inhibitor of metalloproteinase 1 (Timpl), transgelin (Tagln),vimentin (Vin), Fc gamma receptor, cathepsin S (Ctss), and 3 down-regulated genes, i.e., stearyl-CoA desaturase, coagulation factor H (F2), endosulfin alpha (Ensa), were further confirmed by reverse transcription polymerase chain reaction (RT-PCR). These genes may play a role in the response to tissue damage or repair following SCI and characterization of them might be helpful to elucidate the molecular mechanisms of spinal cord injury and regeneration.

Key words: Spinal cord;Gene expression;cDNA microarray;RT-PCR

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引用本文：

肖林, 马振莲, 李欣, 林秋霞, 阙海萍, 刘少君. 应用cDNA微阵列技术筛选大鼠脊髓损伤修复相关基因[J]. 生理学报 2005; 57 (6): .

Xiao Lin, Ma Zhenlian, Li Xin, Lin Qiuxia, Que Haiping, Liu Shaojun. cDNA microarray analysis of spinal cord injury and regeneration related genes in rat. Acta Physiol Sin 2005; 57 (6): (in Chinese with English abstract).