CAJ | 학술논문

目的探讨咪达唑仑对新生大鼠心肌细胞缺氧/复氧时细胞凋亡的影响.方法分离、培养出生2～4 d的Wistar大鼠心肌细胞,将培养融合的心肌细胞随机分为7组(n=7):正常对照组(C组)、缺氧/复氧组(HR组)、外周型苯二氮革类受体(PBR)拮抗剂PK 11195组(P组)、PBR激动剂Ro 5-4864组(R组)、咪达唑仑组(M组)、PK 11195+Ro 5-4864组(PR组)和PK 11195+咪达唑仑组(PM组).HR组缺氧30 min复氧2 h建立缺氧/复氧模型.P组、R组、M组、PR组、PM组在培养皿中分别加入终浓度为10-4mol/L的PK 11195、Ro 5-4864和咪达唑仑及相应混合药物共同孵育,30 min后行缺氧/复氧.复氧2 h时收集细胞,采用HTC-Annexin V/PI双标法检测细胞凋亡情况,计算细胞凋亡指数(AI);Western blot法测定心肌细胞蛋白激酶C(PKc)表达水平;Meta Flour单细胞内钙测定系统测定细胞内钙离子浓度.结果与C组比较,其余各组AI升高,除R组外其余各组细胞内钙离子浓度升高,PKC表达下调(P<0.01);与HR组比较,R组AI和细胞内钙离子浓度降低,PKC表达上调,M组和PM组AI降低(P<0.01);与P组比较,R组AI和细胞内钙离子浓度降低,PKC表达上调(P<0.01),PR组、PM组差异无统计学意义(P>0.05);与R组比较,M组、PR组、PM组Al和细胞内钙离子浓度均升高,PKC表达下调(P<0.01).结论咪达唑仑10-4mol/L可减轻新生大鼠心肌细胞缺氧/复氧时细胞凋亡,其作用机制可能是非PBR依赖性的,且与细胞内钙离子浓度和PKC表达无关.
목적 탐토미체서륜대신생대서심기세포결양/복양시세포조망적영향.방법 분리、배양출생2～4 d적Wistar대서심기세포,장배양융합적심기세포수궤분위7조(n=7):정상대조조(C조)、결양/복양조(HR조)、외주형분이담혁류수체(PBR)길항제PK 11195조(P조)、PBR격동제Ro 5-4864조(R조)、미체서륜조(M조)、PK 11195+Ro 5-4864조(PR조)화PK 11195+미체서륜조(PM조).HR조결양30 min복양2 h건립결양/복양모형.P조、R조、M조、PR조、PM조재배양명중분별가입종농도위10-4mol/L적PK 11195、Ro 5-4864화미체서륜급상응혼합약물공동부육,30 min후행결양/복양.복양2 h시수집세포,채용HTC-Annexin V/PI쌍표법검측세포조망정황,계산세포조망지수(AI);Western blot법측정심기세포단백격매C(PKc)표체수평;Meta Flour단세포내개측정계통측정세포내개리자농도.결과 여C조비교,기여각조AI승고,제R조외기여각조세포내개리자농도승고,PKC표체하조(P<0.01);여HR조비교,R조AI화세포내개리자농도강저,PKC표체상조,M조화PM조AI강저(P<0.01);여P조비교,R조AI화세포내개리자농도강저,PKC표체상조(P<0.01),PR조、PM조차이무통계학의의(P>0.05);여R조비교,M조、PR조、PM조Al화세포내개리자농도균승고,PKC표체하조(P<0.01).결론 미체서륜10-4mol/L가감경신생대서심기세포결양/복양시세포조망,기작용궤제가능시비PBR의뢰성적,차여세포내개리자농도화PKC표체무관.
Objective To investigate the effects of midazolam on hypoxia-reoxygenation induced apoptosis in neonatal rat cardiomyocytes. Methods The myocardial cells of neonatal Wistar rats (2-4 days ) were enzymatically isolated and cultured by using modified Simpson method. Hypoxia-reoxygenation was produced by 30 rain exposure of cells to 99.9% N2 in an air-tight chamber in DMEM liquid culture medium followed by 2 h reoxygenation. The conflnenced ceiLs were divided into 7 groups ( n = 7 each) : group Ⅰcontrol (C) ; groupⅡ hypoxia- reoxygenation (H/R);group Ⅲ PK 11195 [ a peripheral benzodiazepine receptor (PBR) antagonist] (P); group Ⅳ Ro 5-4864 (a PBR agonist) (R); group Ⅴ midazolam (M); group Ⅵ PK 11195 + Ro 5-4864 (PR) and group Ⅶ PK 11195 + midazolam (PM). In group Ⅲ-Ⅵ PK 11195, Ro 5-4864 and midazolam were added to the cells alone or in combination. Their final concentration was 10-4 mol/L. The cells were then incubated for 30 rain before being subjected to H/R. The apoptosis in the cells was detected by flow cytometry. The expression of protein kinase C(PKC) ( by Western blot) and the intracellular calcium concentration ( by Meta Flour system) were determined. Results The apoptosis index (A1) was significantly higher in group Ⅱ-Ⅶ than in control group. The intracellular Ca2+ concentration was significantly higher and the expression of PKC was significantly lower in group H/R, P, M, PR and PM than in control group. The AI and intracellular Ca2+ concentration were significantly lower and the PKC expression was significantly higher in group R than in group H/R. The M was significantly lower in group M and PM than in group H/R. M and intraeellular Ca2+ concentration were significantly lower and PKC expression was significantly higher in group R than in group P. There was no significant difference in M, intracellular Ca2+ concentration and PKC expression between group PB, PM and group P. M and intracellular Ca2+ concentration were significantly higher and PKC expression was significantly lower in group M, PB and PM than in group R. There was no significant difference in M, intracellular Ca2+ concentration and PKC expression between group PM and M and between group PR and PM. Conclusion Midazolam 10-4 mol/L can attenuate the apoptosis of neonatal rat cardiomyocytes induced by hypoxia-reoxygenation. The mechanism is independent of PBR and not related to PKC and intracellular Ca2+ concentration.