Gene expression profiling of cathepsin D, metallothioneins-1 and-2, osteopontin, and tenascin-C in a mouse spinal cord injury model by cDNA microarray analysis
Gene expression profiling of cathepsin D, metallothioneins-1 and-2, osteopontin, and tenascin-C in a mouse spinal cord injury model by cDNA microarray analysis
复制标题
DOI：
10.1007/s00401-004-0926-z
复制
发表时间：
2005-02-01
影响因子：
12.7
通讯作者：
Moriya, H
中科院分区：
文献类型：
--
作者：
Hashimoto, M;Koda, M;Moriya, H
本研究的目的是利用cDNA微阵列来鉴定参与脊髓损伤愈合的新基因。使C57 BL/6小鼠（7-8周，雄性）在T7/8水平经受脊髓压迫损伤（SCI）（20 g，5 min; SCI组）。对于对照组，小鼠仅进行椎板切除术。在第1、3和7天处死小鼠。在每个时间点将从mRNA转录的cDNA与NIA小鼠15 K微阵列杂交。我们发现84个基因显示出显著的表达变化，包括SCI组中比对照组更高和更低的表达水平[使用对数比（基数2）大于1.0或小于-1.0]。选择5个基因，通过实时逆转录（RT）-PCR进行进一步的定量基因表达分析。组织学检查采用原位杂交和荧光免疫组织化学。选择组织蛋白酶D、金属硫蛋白-1（MT-1）、金属硫蛋白-2（MT-2）、骨桥蛋白（OPN）和腱生蛋白-C进行定量和组织学分析。微阵列分析显示，SCI导致OPN和组织蛋白酶D表达在7天的上调，MT-1，MT-2，和tenascin-C表达在1天。生腱蛋白-C在第7天再次上调。这些值与实时RT-PCR分析结果一致。通过原位杂交和荧光免疫组织化学双标记，MT-1，MT-2和tenascin-C的表达，观察在神经元和胶质细胞在1天，而在7天的主要MT-2和tenascin-C的表达被发现在纤维连接蛋白阳性的成纤维细胞。在第3天和第7天，在活化的巨噬细胞/小胶质细胞中观察到主要的组织蛋白酶D和OPN表达。基因表达谱芯片检测的5个基因在脊髓损伤后的表达均表现出时间和空间上的变化。该系统对于鉴定参与SCI反应的基因是潜在有用的。
The purpose of this study was to use a cDNA microarray to identify new genes involved in healing of spinal cord injury. C57BL/6 mice (7-8 weeks, male) were subjected to spinal cord compression injury (SCI) at the T7/8 level (20 g, 5 min; SCI group). For the control group, mice underwent only laminectomy. Mice were killed at 1, 3 and 7 days. cDNA transcribed from mRNA was hybridized to NIA mice 15K microarrays at each time point. We found 84 genes showing significant expressional changes, including higher and lower expression levels in the SCI groups than in the control [more than 1.0 or less than -1.0 using log ratio (base 2)]. Five genes were selected for further quantitative gene expression analysis by real-time reverse transcription (RT)-PCR. For histological examination, we applied in situ hybridization and fluorescence immunohistochemistry. Cathepsin D, metallothionein-1 (MT-1), metallothionein-2 (MT-2), osteopontin (OPN), and tenascin-C were selected for quantitative and histological analysis. Microarray analysis revealed that SCI led to the up-regulation of OPN and cathepsin D expression at 7 days and also of MT-1, MT-2, and tenascin-C expression at 1 day. Tenascin-C was re-up-regulated at 7 days. These values agreed with those of real-time RT-PCR analysis. By double labeling with in situ hybridization and fluorescence immunohistochemistry, MT-1, MT-2 and tenascin-C expression was observed in neurons and glial cells at 1 day, whereas at 7 days the main MT-2 and tenascin-C expression was found in fibronectin-positive fibroblasts. The main cathepsin D and OPN expression was observed in activated macrophages/microglia at 3 and 7 days. The five genes picked up by microarray gene expression profiling were shown to exhibit temporal and spatial changes of expression after SCI. This system is potentially useful for identifying genes that are involved in the response to SCI.