DIFFERENTIALLY EXPRESSED GENES IN ENDOTHELIAL CELLS UNDERGOING APOPTOSIS

大规模DNA测序的最新进展为所有人带来了 人类基因在视野中。阐明哪些基因是最重要的 细胞死亡、增殖和分化等过程仍然存在 这是一项艰巨的任务，需要新的战略和技术。 到目前为止，最有价值的战略之一是识别 这些差异表达的基因在不同的 生理或病理生理学状况。不幸的是，在那里 没有快速、可靠的方法来识别这些基因。 这项研究将扩展我们非常初步的工作，进行大规模的 差异表达基因的分析和评价 在人内皮细胞凋亡过程中的表达。迄今为止，我们 已经设计了一种方案来标记少量的肾上皮细胞 细胞RNA具有很高的比活性，并用它来探测 带有27,648个cDNA阵列的商用膜 其部分序列在表达序列标签中可用 (EST)数据库。使用这一反向的北向分析，我们观察到 组织特异性mRNA的表达模式。总体目标为1) 完整的实验验证了我们的特异性和敏感性 方法和2)利用改进的逆北法进行识别 受刺激的人脐静脉内皮细胞差异表达基因 细胞凋亡。 这项工作的结果对哈佛大学的使命将是重要的 皮肤病研究中心有几个原因。这个项目是 明确专注于大规模差异基因表达 皮肤病领域内的所有研究人员都可以获得分析。 此外，它将使调查人员能够开始识别和获取 在75,000个表达基因中的那些精选基因很可能是 在重要的生物过程中发挥关键作用。最后，通过选择 研究内皮细胞的凋亡，我们将确定 对了解脉管炎、伤口愈合、 自身免疫性疾病、抗磷脂病和其他生物学疾病 皮肤病的重要方面。

Recent progress in large scale DNA sequencing has brought access to all human genes within view. Elucidating which genes are most important in processes such as cell death, proliferation, and differentiation remains a daunting task that will require new strategies and technologies. Among the most valuable strategies to date has been the identification of those genes that are differentially expressed under various physiological or pathophysiological conditions. Unfortunately, there are no rapid, reliable methods to identify these genes. This study will extend our very preliminary work performing large scale differential mRNA expression analysis and evaluate differential gene expression in human endothelial cells undergoing apoptosis. To date, we have devised a protocol to label a small quantity of renal epithelial cell RNA to very high specific activity and used it to probe a commercially available membrane spotted with an array of 27,648 cDNAs whose partial sequences are available in the expressed sequence tags (EST) database. Using this reverse northern analysis we observed a pattern of tissue-specific mRNA expression. The overall goals are to 1) complete experiments validating the specificity and sensitivity of our method and 2) use this improved reverse northern method to identify differentially expressed genes in HUVECs stimulated to undergo apoptosis. The results of this work will be important to the mission of the Harvard Skin Disease Research Center for several reasons. This project is explicitly focused on making large scale differential gene expression analysis available to all investigators within the skin disease field. Further, it will enable investigators to begin to identify and obtain those select genes of the 75,000 expressed genes that are likely to be key players in important biological processes. Finally, by choosing to study apoptosis in endothelial cells, we will identify genes of potential significance for understanding vasculitis, wound healing, autoimmune diseases, anti-phospholipid disease, and other biologically important facets of skin disease.