| Co-Investigator(Kenkyū-buntansha) |
WATANABE Takao Yamagata Univ, Med, Associate Professor, 医学部, 助教授 (60138922)
SHIMAZAKI Yasuhisa Yamagata Univ, Med, Professor, 医学部, 教授 (60116043)
高橋 俊樹 山形大学, 医学部, 講師 (50263257)
箕輪 隆 山形大学, 医学部, 助手 (50292420)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Heat shock protein plays an important role in protecting myocardium from ischemia reperfusion injury. Induction of stress proteins including heat shock protein are reported to occur not only under high temperature but low temperature in microorganism. Recently several studies reported that the mammmarian tissue also express stress proteins under low temperature. Although the topical cooling in open heart surgery is employed for the reduction of basic metabolism, it is conceivable that the low temperature might induce stress protein in the myocardium effective for myocardial preservation. So we studied the protective effect of cold stress on myocardium in ischemia reperfusion injury by the cell culture model of rat H9c2 cell. Method : H9c2 cells were incubated for 24 hours in anoxic chamber followed by normoxic circumstance as a model of ischemia reperfusion injury. Three kinds of thermo-stress, 37℃, 42℃, 4℃ were assigned for H9c2 cell before ischemia. The cellular activity, intracellul
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ar pH, intracellular reactive oxygen species, intracellular glutathione level were studied by loading fluorescent dye calcein-AM, BCECF-AM, H2DCF-DA, Green CMFDA, respectively, for flow cytometer analysis. The influence of Mn-SOD was also studied by mRNA copy count and antisense experiment. Results : The cell viability and intracellular pH were kept well by inducing thermo-stress, 42℃ or 4℃. Mean fluorescent intensity of cells loaded with calcein-AM after ischemia reperfusion was 79% in 37℃ group, but 95% in 42℃ group and 93% in 4℃ group compared to the control (p<0.05, 37℃ vs. 42℃, 4℃). Intracellular reactive oxygen species were increased after ischemia reperfusion, but this increase was marked in 4℃ group. Mean fluorescent intensity of cells loaded with H_2DCFDA was 353% in 37℃ group, 382% in 42℃ group and 528% in 4℃ group (p<0.05, 4℃ vs. 37℃, 4℃ vs. 37℃). The intracellular glutathione level increased slightly, but this change was not significant. Immediate increase of Mn-SOD mRNA level was induced by thermo-stress. Mn-SOD mRNA level increased 4.3 fold 5 minites after 42℃ heat stress and 3.5 fold 15 minites after 4℃ cold stress. The effect of thermo-stress was disappeared in the presence of Mn-SOD antisense oligonucleotide. Conclusions : Not only heat stress but cold stress were effective for protecting cells from ischemia reperfusion injury. Mn-SOD induced by thermo stress may contribute to this protective effect. Less
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