Temporal genomics of vein bypass grafting through oligonucleotide microarray analysis
Temporal genomics of vein bypass grafting through oligonucleotide microarray analysis
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DOI：
10.1016/j.jvs.2003.10.049
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发表时间：
2004-03-01
影响因子：
4.3
通讯作者：
Logerfo, FW
中科院分区：
文献类型：
--
作者：
Kalish, JA;Willis, DJ;Logerfo, FW
目的：自体静脉是小动脉重建的首选导管。尽管具有良好的通畅性，但随着时间的推移，这些导管会发生重塑。本研究的目的是通过微阵列分析来鉴定移植物静脉与对照静脉的时间基因表达。方法：采用犬头静脉旁路移植术（12例）。分别于1、7、14、30天后采集静脉移植物。正常对侧头静脉作为对照。提取总RNA；用分光光度法和凝胶电泳法确定了其数量和质量。Affymetrix U133A基因芯片包含约15,000个基因，用于分析每个时间点的差异基因表达。使用Affymetrix和dChip软件进行统计分析，以确定持续上调和下调的基因。实时、定量逆转录酶聚合酶链反应（qRT-PCR）和免疫组织化学用于验证微阵列数据。结果：统计分析显示，3只动物在不同时间点均有49个基因持续上调，31个基因持续下调。对特定基因进行qRT-PCR定量评估信使RNA表达，以验证微阵列数据。免疫组织化学定性评估蛋白表达进一步验证。dChip的分层聚类鉴定出具有相似时间或功能表达模式的其他基因。结论：这是首次使用微阵列分析和验证性qKT-PCR来鉴定静脉旁路移植术后改变的基因。寡核微粒微阵列和分层聚类是产生假设的有力工具，为进一步研究移植物重塑中的基因表达奠定了基础。最终，鉴定差异基因表达的时间序列不仅可以为静脉移植物重塑的分子机制提供更好的见解，还可以为导致内膜增生的途径提供更好的见解。
Objective: Autologous vein is the conduit of choice for small artery reconstruction. Despite excellent patency, these conduits undergo remodeling over time. The purpose of this study was to identify temporal gene expression in vein grafts versus control veins through microarray analysis.Method: Ceplialic vein grafts (n = 12) were used to bypass femoral arteries in canines. Vein grafts were harvested after 1, 7, 14, and 30 days. Normal contralateral cephalic vein served as control. Total RNA was isolated; its quantity and quality were confirmed with spectrophotometry and gel electrophores is. Affymetrix U133A GeneChips, comprising approximately 15,000 genes, were used to analyze differential gene expression at each time point. Statistical analysis was performed with Affymetrix and dChip software to identify consistently upregulated and downregulated genes. Real-time, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to validate microarray data.Results: Statistical analysis revealed that 49 genes were consistently upregulated and 31 genes were consistently downregulated in all three animals at various time points. qRT-PCR to quantitatively assess messenger RNA expression was performed on specific genes to validate the microarray data. Immuntohistochemistry to qualitatively assess protein expression was used for further validation. Hierarchical clustering with dChip identified additional genes with similar temporal or functional expression patterns.Conclusions: This is the first study to use microarray analysis with confirmatory qKT-PCR to identify altered genes after vein bypass grafting. Oligonucleoticle microarrays and hierarchical clustering are powerful tools to generate hypotheses as the basis for additional research on gene expression in vein graft remodeling. Ultimately, identification of a temporal sequence of differential gene expression may provide insights not preferred into the molecular mechanisms of vein graft remodeling, but also into the pathways leading to intimal hyperplasia.