摘要
目的 观察体外培养心肌急性缺氧条件下环氧化酶-2（COX2）抑制剂与细胞凋亡间的关系.方法 分离、培养Wistar大鼠心肌细胞.以第4～6代心肌细胞24份样本（每份1×10^5个细胞）随机分为正常对照组、单纯缺氧组、缺氧＋COX-2抑制剂（NS-398,20 μmol/L）组及缺氧＋阿司匹林（100 μg/L）组.缺氧前30 min加入干预药物或等量二甲亚砜.以蛋白质免疫印迹法检测心肌细胞中COX-1/2表达;流式细胞仪检测心肌细胞凋亡率;放射免疫法（RIA）检测心肌细胞培养液中6-酮-前列腺素F1α（6-ketoPGF1α）、血栓素B2（TXB2）含量.结果 各组心肌细胞中COX-1表达差异无显著性;COX2表达均较正常对照组增高,阿司匹林与NS-398均不抑制COX2表达.心肌细胞凋亡率由高到低依次为缺氧＋NS-398组、缺氧＋阿司匹林组、单纯缺氧组和正常对照组;缺氧＋NS-398组与其他各组间比较差异均有显著性（P均〈0.05）.各组心肌细胞培养液内TXB2水平由低到高依次为正常对照组、缺氧＋NS-398组、缺氧＋阿司匹林组和单纯缺氧组;缺氧＋NS-398组与单纯缺氧组比较差异有显著性（P〈0.05）;6-keto-PGF1α水平由低到高依次为正常对照组、缺氧＋NS-398组、缺氧＋阿司匹林组和单纯缺氧组;缺氧＋NS-398组与单纯缺氧组比较差异有显著性（P〈0.05）;TXB2/6-keto-PGF1α比值差异无显著性.结论 急性缺氧可直接诱导培养心肌细胞表达COX-2,而COX-1表达不增加,缺氧使培养液内COX-2作用产物TXB2、6keto-PGF1α水平升高,此效应无中性粒细胞、巨噬细胞等炎症细胞参与.COX-2抑制剂NS-398可降低缺氧心肌培养液内TXB2、6-keto-PGF1α升高程度,但不改变TXB2/6keto-PGF1α比值.急性缺氧可直接诱导心肌细胞凋亡,NS398可显著增加缺氧培养心肌凋亡率,此效应不需要其他组织细胞参与.
Objective To observe the effects of cyclooxygenase （COX）- 2 inhibitor on apoptosis of hypoxic myocardial cells in vitro. Methods The myocardial cells were obtained from new-born Wistar rats, and were dispersed to single cell with trypsin. The cells in 4th - 6th passages were randomly divided into 24 samples （every sample contained 1×10^5 cells）： normal control group, hypoxic myocardial cells control group ; hypoxic myocardial cells ＋ NS - 398 （20μmol/L ） and hypoxic myocardial cells ＋ aspirin （100μg/L）. During culture, oxygen was replaced by N2, and the cells were cultivated in 5% CO2＋95% N2 at 37℃ for 6 hours. Either interventional medicine or same amount of dimethyl sulphoxide （menstruum） was added to the cells 30 minutes before hypoxia. The expression of COX - 1 and COX - 2 in cultured myocardial cells in vitro was examined with Western blot. The apoptosis percentage of cells in each group was examined with flow cytometry. After centrifuging the culture medium under low temperature, 6 -keto-prostaglandin F1α. （6- keto- PGF1α,） and thromboxane B2 （TXB2） were determined with radioimmunoassay （RIA）. Results There was no significant difference in the expression of COX- 1 among the groups. The expression of COX - 2 was higher in all acute hypoxic myocardial cells groups compared with the control group. Neither aspirin nor NS - 392 inhibited the expression of COX - 2. The positive percentage of apoptosis of cultured myocardial ceils ranked as follow： hypoxic＋NS- 398 group, hypoxic ＋aspirin group, hypoxic control group and normal control group. The differences of apoptosis rate between hypoxic ＋ NS - 398 group and other groups were significant （all P〈0. 05）. The levels of TXB2 in each group of cell medium ranked in the following order： normal control group, hypoxic ＋NS - 398 group, hypoxic ＋aspirin group and hypoxic control group. The difference of TXB2 level between hypoxic＋NS - 398 group and hypoxic control group was significant （P〈0.05）. The levels of 6 - keto - PGF1α in each culture medium ranked in the following order ： normal control group, hypoxic ＋aspirin group, hypoxic＋NS- 398 group and hypoxic control group. The difference between hypoxic＋NS- 398 group and hypoxic control group was significant（P〈0.05）. The ratio of TXB2/6 - keto - PGF1α showed no significant difference （P〉0.05 ） among groups. Conclusion The results suggest that acute hypoxia could directly induce cultured myocardial cells to express COX - 2, but do not effect expression of COX - 1. Hypoxia could elevate the level of 6 - keto - PGF1α and TXB2 in culture medium. COX- 2 inhibitor （NS- 398） could lessen the elevation, but it could not change the ratio between TXB2 and 6- keto PGF1α. NS- 398 could increase apoptosis percentage of hypoxic myocardial cells in vitro, and the effect is independent of other inflammatory cells.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2006年第4期229-232,共4页
Chinese Critical Care Medicine
基金
全军"十五"科研基金面上项目(01MA098)
关键词
环氧化酶-2抑制剂
心肌
缺氧
细胞凋亡
cyclooxygenase - 2 inhibitor
myocardial cell
hypoxia
apoptosis
