Ischemia-responsive Protein(irp94) Gene Expression in a Neuronal Cell Culture Model of Ischemia.
- Author:
Seung Hwan KIM
1
;
Young Mo YANG
;
Young Rok HA
;
Sung Pil CHUNG
;
In Sool YOO
;
In Byung KIM
Author Information
1. Department of Emergency Medicine, Chungnam National University Hospital, Korea. emdfire@hanmail.net
- Publication Type:In Vitro ; Original Article
- Keywords:
irp94;
Gene expression;
Ischemic neuronal injury
- MeSH:
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
- From:Journal of the Korean Society of Emergency Medicine
2001;12(3):201-206
- CountryRepublic of Korea
- Language:Korean
-
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.
