Gene expression profiling of thermal-induced injury: A drug discovery and development tool for evaluating potential cytoprotective agents for the amelioration of thermal and laser injury to the eye.

摘要

Due to the proliferation of novel applications of laser technology in medicine, research and the military, instances of laser injury, particularly to the eye, have considerably risen in the past three decades. This injury is predominantly a thermal injury produced by a short pulse of relatively high temperature that involves irreversible damage at the burn site, and a secondary injury that results from the inflammatory response initiated by thermally injured cells. An uncontrolled inflammatory response often causes permanent and irreversible damage to both normal and initially damaged yet viable cells. If caught in time though, much of the secondary damage may be prevented or ameliorated.; The objective of this research was to understand the genetic response of human cells to thermal injury, such as produced by lasers, and then to use this information to develop appropriate therapeutic treatment. The specific aims proposed were to: (1) establish an in vitro model for thermal injury using retinal pigment epithelial cells (ARPE-19), (2) determine the pattern of gene expression in response to thermal injury using gene expression profiling, (3) select potentially beneficial therapeutics based on their ability to affect metabolic pathways identified by gene expression profiling, and to (4) evaluate the effects of candidate therapeutics on thermal induced injury in vitro.; The extent of injury and the effectiveness of cytoprotective were evaluated by observing changes in cell viability, nitrite production, and cytokine and stress protein synthesis. Gene regulation of cytokines, transcription factors, stress response and protective proteins such as IL-1, IL-8, TNF-α, HSP70, GADD153 and NFκB were determined by gene microarrays, and compared by RT-PCR, ELISA, Western blotting, and immunohistochemistry.; By observing the alterations in inflammatory response to thermal stress, we may better understand the benefits and targets of drug treatment for secondary damage associated with laser injury. In the near future, genetic profiles can be created for nearly any cell type or disease state, giving a fingerprint of sorts. As we gather more knowledge of how genes are regulated and function, we can use these “fingerprints” to logically aid in drug discovery and development.