Hsp70
Assessment of
laser-induced tissue damage is not complete without an investigation into the
resulting cellular and molecular changes. In the past, tissue damage was
quantified macroscopically by visual effects such as tissue mass removal,
carbonization and melting. Microscopically, assessment of tissue damage has
been typically limited to histological analysis of excised tissue samples. In
this research, we used heat shock protein (hsp70) transcription to track
cellular response to laser-induced injury. A stable cell line (NIH-3T3) was
generated containing the firefly luciferase (luc) reporter gene attached to the hsp
promoter (murine hsp70a1). After thermal injury with
a pulsed holmium-yttrium aluminum garnet laser (lambda = 2.1 microm, taup = 250 micros, 30
pulses, 3 Hz), luciferase is produced on hsp70
activation and emits broad-spectrum bioluminescence over a range of 500-700 nm,
with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h
after treatment and usually peaks at 8-12 h depending on the severity of heat
shock. The luminescence was quantified in live cells using bioluminescence
imaging. A minimum pulse energy (65 mJ/pulse [total
energy 1.95 J; total radiant exposure = 6 J/cm2]) was needed to activate the
hsp70 response, and a higher energy (103 mJ/pulse
[total energy 3.09 J; total radiant exposure = 9.6 J/cm2]) was associated with
a reduction in hsp70 response and cell death. Bioluminescence levels correlated
well with actual hsp70 protein concentrations as determined by enzyme-linked immunosorbent assay. Photon counts were normalized to the
percentage of live cells by means of a flow cytometry
cell viability assay. Within a relatively small range between a lower
activation threshold and an upper threshold that leads to cell death, the hsp70
response followed an Arrhenius relationship when
constant-temperature water bath and laser experiments were carried out.
PMID: 14974719 [PubMed - in process]