摘要
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2～4d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PK11195组（P组）、PBR激动剂Ro5—4864组（R组）、咪达唑仑组（M组）、PK11195＋Ro5-4864组（PR组）和PK11195＋咪达唑仑组（PM组）。HR组缺氧30min复氧2h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PK11195、Ro5-4864和咪达唑仑及相应混合药物共同孵育，30min后行缺氧／复氧。复氧2h时收集细胞，采用FITC—AnnexinV／PI双标法检测细胞凋亡情况，计算细胞凋亡指数（AI）；Westernblot法测定心肌细胞蛋白激酶C（PKC）表达水平；MetaHour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AI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。
Objective To investigate the effects of midazolam on hypoxia-reoxygenation induced apoptosis in neonatal rat cardiomyoeytes. 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 min exposure of cells to 99.9% N2 in an air-tight chamber in DMEM liquid culture medium followed by 2 h reoxygenation. The confluenced cells were divided into 7 groups （ n = 7 each） ： group Ⅰ control （C） ; group Ⅱ hypoxia-reoxygenation （H/R） ; group Ⅲ PK 11195 [ a peripheral benzodiazepine receptor （PBR） antagonist] （P） ; group IV Ro 5-4864 （a PBR agonist） （R）; groupV midazolam （M）; group Ⅵ PK 11195 ＋ Ro 5-4864 （PR） and group ⅦPK 11195 ＋ midazolam （PM）. In group Ⅲ-Ⅶ 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 min 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 （AI） was significantly higher in group Ⅲ-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.Tbe AI was significantly lower in group M and PM than in group H/R. AI and intracellular Ca2＋ concentration were signifieandy lower and PKC expression was significantly higher in group R than in group P. There was no significant difference in AI, intracellular Ca2＋ concentration and PKC expression between group PR, PM and group P. AI and intracellular Ca2＋ concentration
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2008年第8期746-749,共4页
Chinese Journal of Anesthesiology
基金
辽宁省教育厅高等学校科学技术研究基金资助项目（2004D181）
关键词
咪达唑仑
再灌注损伤
心肌
细胞凋亡
Midazolam
Myocardial reperfusion injury
Myocardium
Apoptosis
