CAJ | 학술논문

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、400 μg/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400 μg/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、400 μg/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400 μg/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、400 μg/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200 μg/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200 μg/ml组药物干预24h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400 μg/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400 μg/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、400 μg/ml组细胞的ADP/ATP比值明显高于对照组(1.14 ±0.11、1.31±0.18比0.98±0.09,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.
목적 탐토중조인혈관내피억소(은도)심장독성작용적파표급작용궤제. 방법 이H9c2심기세포위관찰대상,진행이하실험:(1)장H9c2세포분위대조조(불급여약물간예)화은도100、200、400 μg/ml조(가입상응농도약물배양24、48 h),용류식세포술검측각조각시점세포조망솔;(2)장H9c2세포분위대조조화은도400 μg/ml간예24 h실험조,용투사전경관찰세포초미결구개변;(3)장H9c2세포분위대조조화은도100、200、400 μg/ml조(가입상응농도약물배양18 h),응용JC-1형광탐침검측세포선립체막전위;(4)장H9c2세포분위대조조화은도400 μg/ml간예24 h실험조,용세포면역화학방법관찰세포색소C (Cyt-C)석방정황;(5)장H9c2세포분위대조조화은도100、200、400 μg/ml조(가입상응농도약물배양24 h),용화학발광법검측각조세포적ADP/ATP비치. 결과 (1)은도200 μg/ml조재약물간예24 h、은도400μg/ml조재약물간예24、48 h후,세포조망솔균명현고우대조조[24 h:(16.34±3.72)％、(27.03 ±3.91)％비(6.99±1.72)％;48 h:(24.89 ±4.77)％비(6.44±1.81)％,균P＜0.01];은도200 μg/ml조약물간예24h후적세포조망솔고우약물간예48 h[(16.34 +3.72％)비(11.34±3.09)％,P＜0.01].(2)대조조세포초미결구정상;은도400 μg/ml간예24 h실험조세포핵고축쇄렬、염색질괴취,세포내공포증다,내질망확장,선립체종창,출현조망소체.(3)여대조조상비,불동제량은도간예조세포선립체과막전위거겁화정도수은도농도증가이하강.(4)대조조세포Cyt-C주요분포우선립체,은도400 μg/ml간예24 h실험조세포Cyt-C종선립체석방지포질.(5)은도200、400 μg/ml조세포적ADP/ATP비치명현고우대조조(1.14 ±0.11、1.31±0.18비0.98±0.09,균P＜0.01). 결론 심기세포선립체가능시은도심장독성작용적파표,경선립체의뢰성도경유도적심기세포조망시은도치심기손상적궤제지일.
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 μg/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 μg/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 groups including 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 cocultured for 24 h group and the 400 μg/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 ＜0.01].The apoptotic rate of the 200 μg/ml endostar co-cultured 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 H9c2 cells' ultrastructure of control group presented normal.The H9c2 cells in the group of 400 μg/ml endostar co-cultured for 24 h showed pyknotic and fragmental in nuclei,the massed chromatin,intracellular vacuoles,dilation of endoplasmic reticulum,mitochondrial swelling,and appearance of apoptotic body.(3) Compared with the control group,the degree of depolarization of mitochondrial transmembrane potential decreased following of the increasing concentration of endostar in 100,200,and 400 μg/rnl endostar intervened groups.(4) Cytochrome C of the H9c2 cells in the control group mainly distributed in the mitochondria.Cytochrome C of the H9c2 cells in the group of 400 μg/ml endostar cocultured for 24 h were released from mitochondria to the cytoplasm.(5) ADP/ATP ratio of H9c2 cells in the 200 μg and 400 μg/ml endostar groups were significantly higher than those in the control group (1.14 ±0.11,1.31 ± 0.18 vs.0.98 ± 0.09,all P ＜ 0.01).Conclusions Cardiomyocyte mitochondria may be the target of the cardiotoxicity of endostar.The cardiomyocyte apoptosis evoked via the mitochondrial-dependent pathway is one of the mechanism of myocardial damage.