Microarray Analysis of Cellular Thermotolerance
Microarray Analysis of Cellular Thermotolerance
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DOI：
10.1002/lsm.20983
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发表时间：
2010-12-01
影响因子：
2.4
通讯作者：
Jansen, E. Duco
中科院分区：
文献类型：
--
作者：
Beckham, Josh T.;Wilmink, Gerald J.;Jansen, E. Duco
背景和目的：之前，我们已经证明43℃预处理可以提供对后续更严重的45℃热应激的耐热性。使用缺乏Hsp70基因的细胞，我们还证明在缺乏Hsp70蛋白的情况下仍有一定的耐热性。本研究的目的是通过测量细胞在加热后2天的活力和增殖来确定哪些基因在耐热性中起作用。具体来说，我们想要了解在缺乏HSP70的情况下，哪些途径可能负责保护细胞。研究设计/材料和方法：将含Hsp70和不含Hsp70的小鼠胚胎成纤维细胞（MEF（+/+）和MEF(-/-)）在43℃的恒温水浴中暴露30分钟。回收3小时后，使用带有DNAse处理的MicroRNeasy试剂盒从三个加热的样品和三个未处理的对照中收集RNA。RNA质量由安捷伦生物分析仪验证。然后将RNA转化为cDNA，并杂交到Affymetrix基因表达DNA微阵列中。与未加热的对照相比，显示两倍变化（向上或向下）的基因使用genesspring软件进行t检验，在P < 0.05的阈值下进行显著性过滤。数据经qRT-PCR验证。然后根据它们的本体对基因进行分类。结果：虽然许多基因类似上调，但细胞类型之间的主要区别是转录因子和核酸结合蛋白的增加。几个已知参与热反应的基因（Hsp70, Hsp40, Hsp110, Hsp25, Atf3）上调了两倍以上，然而，另一个被充分研究的热反应基因Hsp90在这些条件下仅增加了1.5倍，尽管它在耐热性中起作用。结论：这些数据为进一步研究激光照射前处理方案提供了遗传途径。中国激光医学杂志，2010。(C) 2010 Wiley-Liss, Inc。
Background and Objectives: Previously, we have shown that a 43 degrees C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45 degrees C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70.Study Design/Materials and Methods: Murine embryonic fibroblast cells with and with out Hsp70(MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43 degrees C for 30 minutes in a constant temperature water bath. After 3 hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a two-fold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P < 0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology.Results: While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance.Conclusions: The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation. Lasers Surg. Med. 42:752-765, 2010. (C) 2010 Wiley-Liss, Inc.