摘要
目的 建立缺氧诱导体外培养的成人心肌细胞凋亡情况。方法 用胰蛋白酶和胶原酶进行消化 ,用差速黏附贴壁法、Brdu加入和人为破坏成纤维细胞法纯化心肌细胞 ,用IMDM培养基培养 ,4～ 6d后将体外培养的成人心肌细胞置于 3 %氧气、92 %氮气及 5 %二氧化碳孵箱中 ,6、12、2 4、48h缺氧后再恢复正常条件培养 4h ,造成缺氧损伤所致细胞凋亡模型 ,分别用倒置显微镜、电镜、TUNEL法技术检测凋亡发生的情况。结果 心肌细胞经历缺氧损伤后 ,倒置显微镜、电镜、TUNEL染色均观察到凋亡阳性细胞。TUNEL法定量检测 ,心肌细胞缺氧培养 6、12、2 4、48h后 ,其凋亡率分别为 :( 3 3± 0 9) %、( 8 3± 1 8) %、( 16 1± 2 6) %和( 19 4± 2 3 ) %。结论 心肌细胞凋亡率随着缺氧时间的延长而增高 。
Objective To establish a model for hypoxia-induced apoptosis of human adult cardiomyocytes in vitro. Methods The human adult heart cells of ventricular muscle were isolated by the digestion of trypsin and collagenase, cultured in Iscove's modified Dulbecco's medium (IMDM), and purified by means of differential attachment technique, adding Brdu and slicing fibroblasts by sterile needle. After 4 to 6 days, the cardiomyocytes were cultured in an incubator containing 3% O 2, 92% N 2 and 5% CO 2 for 6, 12, 24 and 48h to form hypoxia injury for cells. Morphological changes of apoptotic cardiomyocytes were measured by invertion microscopy and electronic microscopy. Apoptotic rate was measured by TUNEL staining. Results The cardiomyocytes occurred opoptosis after hypoxia injury, apoptotic rate of which at 6, 12, 24 and 48h after hypoxia was (3 3±0 9)%, (8 3±1 8)%, (16 1±2 6)% and (19 4±2 3)% respectively. Conclusion The apoptotic rate of cardiomyocytes increased with time of hypoxia lengthened. The model for hypoxia-induced apoptosis of human adult cardiomyocytes was successful and reliable.
出处
《中国医师杂志》
CAS
2004年第7期865-867,共3页
Journal of Chinese Physician
基金
国家自然科学基金资助项目 (No.30 1 0 0 0 69)
