J Korean Soc Emerg Med.  2001 Sep;12(3):201-206.

Ischemia-responsive Protein(irp94) Gene Expression in a Neuronal Cell Culture Model of Ischemia

• Kim SH
• Yang YM
• Ha YR
• Chung SP
• Yoo IS
• Kim IB

Abstract

BACKGROUND: The ischemia responsive protein 94 kDa(irp94) gene belongs to the heat shock protein 110 family and was isolated in 1999 from rat brain by transiently induced forebrain ischemia. The PC12 cell is the pheochromocytoma cell line of rat, which is differentiated to a sympathetic neuron-like cell by the stimulation of a nerve growth factor. This study is to determine whether irp94 is expressed when an ischemia-like condition is induced by ATP depletion in cultured PC12 cells in vitro.
METHODS
PC12 cells were maintained as monolayer cultures in RPMI-1640 medium(Sigma) supplemented with 10% horse serum, 5% fetal bovine serum, 5 mg/ml transferrin, and 1 mg/ml insulin in a humidified 5% CO2 incubator at 37degrees C. The ATP depleting agent antimycin A was added at concentrations of 1, 2.5, and 5 microM to simulate ischemia, and 10 microgram/ml of tunicamycin, which is expected to express heat shock protein maximally, was used as a positive control. The cells were harvested after a 60-minute incubation, and the total RNA was extracted. The reverse transcription polymerase chain reaction(RT-PCR) was performed to use 501 bp irp94 cDNA as a molecular probe, and the expression of irp94 mRNA was analyzed by northern blotting.
RESULTS
The irp94 mRNA expression was enhanced, compared to the negative control group, as the concentration of antimycin A was increased.
CONCLUSION
This study suggests that irp94 mRNA expression is enhanced as the severity of ischemia is increased. Thus, it is possible to investigate the mechanism of ischemic neuronal injury indirectly by using this in-vitro model of neuronal ischemia.

Keyword

irp94; Gene expression; Ischemic neuronal injury

MeSH Terms

Adenosine Triphosphate
Animals
Antimycin A
Blotting, Northern
Brain
Cell Culture Techniques*
DNA, Complementary
Gene Expression*
Heat-Shock Proteins
Horses
HSP110 Heat-Shock Proteins
Humans
Incubators
Insulin
Ischemia*
Molecular Probes
Nerve Growth Factor
Neurons*
PC12 Cells
Prosencephalon
Rats
Reverse Transcription
RNA
RNA, Messenger
Transferrin
Tunicamycin
Adenosine Triphosphate
Antimycin A
DNA, Complementary
HSP110 Heat-Shock Proteins
Heat-Shock Proteins
Insulin
Molecular Probes
Nerve Growth Factor
RNA
RNA, Messenger
Transferrin
Tunicamycin