Identification of Aeromonas hydrophila cytotoxic enterotoxininduced genes in macrophages using microarrays
Identification of Aeromonas hydrophila cytotoxic enterotoxininduced genes in macrophages using microarrays
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DOI：
10.1074/jbc.m305788200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Chopra, AK
中科院分区：
文献类型：
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作者：
Galindo, CL;Sha, J;Chopra, AK
嗜水气单胞菌的细胞毒性肠毒素（Act）具有多种生物活性，并且它在宿主中诱导炎症反应。在这项研究中，我们使用微阵列来获得一个全球性的和分子的观点的细胞转录反应的行为，并确定重要的基因上调这种毒素。在0、2和12小时从ACT处理的巨噬细胞中分离总RNA，并应用于Affytron MGU 74阵列，并使用多分析方法处理数据以鉴定可能在ACT诱发的炎症过程中至关重要的基因。76个基因显著且一致地上调。这些基因中有许多与免疫相关，有几个是转录因子、粘附分子和细胞因子。此外，我们确定了几个凋亡相关的基因，显着上调ACT处理的巨噬细胞。ACT诱导的巨噬细胞凋亡通过Annexin V染色和DNA梯状条带证实。定量逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附试验被用来验证一些炎症和前列腺增生相关基因的表达增加的微阵列分析确定。为了进一步证实ACT诱导的基因表达增加，还将实时RT-PCR用于所选基因。总之，阵列数据第一次提供了ACT介导的信号转导的全局视图，并清楚地证明了在分子水平上由该毒素在宿主细胞中介导的炎症反应和细胞凋亡。
A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, and it induces an inflammatory response in the host. In this study, we used microarrays to gain a global and molecular view of the cellular transcriptional responses to Act and to identify important genes up-regulated by this toxin. Total RNA was isolated at 0, 2, and 12 h from Act-treated macrophages and applied to Affymetrix MGU74 arrays, and the data were processed using a multi-analysis approach to identify genes that might be critical in the inflammatory process evoked by Act. Seventy-six genes were significantly and consistently up-regulated. Many of these genes were immune-related, and several were transcription factors, adhesion molecules, and cytokines. Additionally, we identified several apoptosis-associated genes that were significantly up-regulated in Act-treated macrophages. Act-induced apoptosis of macrophages was confirmed by annexin V staining and DNA laddering. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to verify increased expression of some inflammatory and apoptosis-associated genes identified by the microarray analysis. To further confirm Act-induced increases in gene expression, real-time RT-PCR was also used for selected genes. Taken together, the array data provided for the first time a global view of Act-mediated signal transduction and clearly demonstrated an inflammatory response and apoptosis mediated by this toxin in host cells at the molecular level.