摘要
目的探讨重组人血管内皮抑素(恩度)心脏毒性作用的靶标及作用机制。方法以H9c2心肌细胞为观察对象,进行以下实验:(1)将H9c2细胞分为对照组(不给予药物干预)和恩度100、200、400μg/ml组(加入相应浓度药物培养24、48 h),用流式细胞术检测各组各时点细胞凋亡率;(2)将H9c2细胞分为对照组和恩度400μg/ml干预24 h实验组,用透射电镜观察细胞超微结构改变;(3)将H9c2细胞分为对照组和恩度100、200、400μg/ml组(加入相应浓度药物培养18 h),应用JC-1荧光探针检测细胞线粒体膜电位;(4)将H9c2细胞分为对照组和恩度400μg/ml干预24 h实验组,用细胞免疫化学方法观察细胞色素C(Cyt-C)释放情况;(5)将H9c2细胞分为对照组和恩度100、200、400μg/ml组(加入相应浓度药物培养24 h),用化学发光法检测各组细胞的ADP/ATP比值。结果(1)恩度200μg/ml组在药物干预24 h、恩度400μg/ml组在药物干预24、48 h后,细胞凋亡率均明显高于对照组[24 h:(16.34±3.72)%、(27.03±3.91)%比(6.99±1.72)%;48 h:(24.89±4.77)%比(6.44±1.81)%,均P<0.01];恩度200μg/ml组药物干预24 h后的细胞凋亡率高于药物干预48 h[(16.34+3.72%)比(11.34±3.09)%,P<0.01]。(2)对照组细胞超微结构正常;恩度400μg/ml干预24 h实验组细胞核固缩碎裂、染色质块聚,细胞内空泡增多,内质网扩张,线粒体肿胀,出现凋亡小体。(3)与对照组相比,不同剂量恩度干预组细胞线粒体跨膜电位去极化程度随恩度浓度增加而下降。(4)对照组细胞Cyt-C主要分布于线粒体,恩度400μg/ml干预24 h实验组细胞Cyt-C从线粒体释放至胞质。(5)恩度200、400μg/ml组细胞的ADP/ATP比值明显高于对照组(1.14±0.11、1.31±O.18比0.98±O.09,均P<0.01)。结论心肌细胞线粒体可能是恩度心脏毒性作用的靶标,经线粒体依赖性途径诱导的心肌细胞凋亡是恩度致心肌损伤的机制之一。
Objective To explore the target and mechanisms of the cardiotoxicity of recombinant human endostatin （endostar）. Methods The myocardial cell line H9c2 was used as subjects and the following tests were performed. （1） H9c2 cells were divided into the control group （without drug intervention）, intervened groups including 100, 200, and 400 μg/ml endostar co-cultured for 24 h or 48 h, respectively. The apoptosis rate of H9c2 cells in different groups were detected by flow cytometry. （2） The H9c2 cells were divided into the control group and the intervened group of 400 txg/ml endostar co-cultured for 24 h. The changes of ultrastructure of cells were observed by transmission electron microscope. （3） The H9c2 cells were divided into the control group and the intervened groups including 100, 200, and 400 t^g/ml endostar co-cultured for 18 h, respectively. The mitochondrial membrane potential was recorded by JC-1 fluorescence probe. （4） The H9c2 cells were divided into the control group and the intervened group of 400 μg/ml endostar co-cultured for 24 h. The release condition of cytochrome C were observed by method of immunocytochemistry. （5） The H9c2 cells were divided into the control group and the intervened groupsincluding 100, 200, and 400 μg/ml endostar co-cultured for 24 h, respectively. The ADP/ATP ratio was detected by the method of chemiluminescence. Results （ 1 ） The apoptotic rate of 200 μg/ml endostar co- cultured for 24 h group and the 400 ~g/ml endostar co-cultured for 24 h and 48 h groups were higher than that in the control group [ 24 h ： （ 16.34 ＋ 3.72） %, （27.03 -＋ 3.91 ） % vs. （6.99 ＋ 1.72 ） % ; 48 h ： （24.89 ＋ 4. 77）% vs. （6.44 ＋ 1.81 ）% ; all P 〈0.011- The apoptotic rate of the 200 p^g/ml endostar co-cnltured for 24 h group was higher than that in the endostar co-cultured for 48 h group[ （16.34 ＋3.72）% vs. （11.34 -＋ 3.09） %, P 〈 0.01 ]. （2） The H9e2 cells＇ ultrastructure of control group presented normal. The H
出处
《药物不良反应杂志》
CSCD
2013年第6期336-341,共6页
Adverse Drug Reactions Journal
基金
国家自然科学基金(81202806)
