期刊
LASERS IN SURGERY AND MEDICINE
卷 42, 期 10, 页码 752-765出版社
WILEY
DOI: 10.1002/lsm.20983
关键词
heat shock protein; HSP70; pretreatment; thermal stress; gene expression analysis; microarray; RT-PCR; cell culture
资金
- ASLMS (American Society for Laser Medicine Surgery)
- DOD/AFOSR [F49620-01-1-4029]
- Vanderbilt Medical Center
- Vanderbilt Ingram Cancer Center [P30 CA68485]
- Vanderbilt Digestive Disease Center [P30 DK58404]
- Vanderbilt Vision Center [P30 EY08126]
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.
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