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模拟缺血/再灌注诱导小鼠心肌细胞凋亡模型的构建及其生化特征(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2008年第3期
页码:
259-263
栏目:
基础研究
出版日期:
2008-05-20

文章信息/Info

Title:
Establishment and biochemical characters of mouse cardiomyocytes apoptos is induced by simulated ischemia/reperfusion
作者:
曹亚南1刘安恒12张卫卫1师堂旺1刘艳1王晓明1
第四军医大学西京医院: 1.老年病科,2.心脏内科,陕西 西安 710032
Author(s):
CAO Yanan1 LIU Anheng12 ZHANG Weiwei1 SHI Tangwang1 LIU Yan1 WANG Xiaoming1
1.Department of Geriatrics, 2.Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi, China
关键词:
心肌细胞缺血/再灌注凋亡
Keywords:
cardiomyocytes apoptosis ischemia/reperfusion
分类号:
R542.2
DOI:
-
文献标识码:
A
摘要:
目的 建立稳定的缺血/再灌注(I/R) 诱导小鼠心肌细胞凋亡模型并观察其生化特征。方法 原代培养的小鼠心肌细胞在模拟I/R条件下,观察细胞存活率、乳酸脱氢酶(LDH)水平,半胱天冬蛋白酶(Caspase)3水平、DNALadder现象。结果 ①不同的缺血和再灌注时间,均能够造成心肌细胞的损伤,随着缺血时间的延长细胞损伤以坏死性死亡为主要方式,而再灌注损伤主要方式是细胞凋亡。②在缺血6 h再灌注96 h过程中发现:心肌细胞出现明显的皱缩、胞核染色质断裂、聚集、固缩等典型的凋亡形态学变化;I/R组的细胞存活率是522%,与正常对照组100%之间有显著性差异(n=20,P<001);在缺血期Caspase3活性和LDH释放水平呈现线形增加,而活性氧(ROS)变化不大;在再灌注期Caspase3活性和ROS于24 h达峰值,96 h后逐步恢复接近正常水平;LDH释放水平缓慢增加,96 h后仅为35%。96 h后可见明显的DNALadder现象。结论 模拟缺血6 h和再灌注96 h构建了稳定的小鼠心肌细胞凋亡模型,再灌注损伤是形成凋亡性死亡的主要因素。
Abstract:
AIM To establish a stabilized model of mouse cardiomyocyte apoptosis induced by simulated ischemia/reperfusion (I/R) and to explore the biochemical characters. METHODS Neonatal mouse cardiomyocytes were cultured and treated with simulated I/R. DNA fragmentations, cysteine aspartate specific proteinase (Caspase)3, lactate dehydrogenase (LDH) activity, cell viability and cell membrane integrity were observed. RESULTS ①Not only ischemia but also reperfusion with difference times resulted in cardiomyocyte injure and the main way of cardiomyocyte death was necrosis during ischemia processes but apoptosis during reperfusion processes. ②Apoptotic cardiomyocytes model was successfully induced by 6 hours’ ischemia, followed by 96 hours’ reperfusion. Striking morphological changes, including the shrinkage of cells, membrane blebbing, chromatin condensation and cellular DNA breakdown, were observed. Compared on The cell viability in I/R group and control group were 522% and 100%, with significant differences (n=20, P<001) after 6 hours’ ischemia and 96 hours’ reperfusion. The Caspase3 activity and release level of LDH were linearly increased with ischemic times, but no significant changes were observed in reactive oxygen species (ROS)levels. During reperfusion processes, the Caspase3 activity and ROS levels reached peak after 24 hours and recovered normal levels gradually. Increasing release level of LDH became slower and the typical DNA ladders appeared after 96 hours’ reperfusion. CONCLUSION Simulated 6 hours’ ischemia and 96 hours’ reperfusion are the optimal conditions to induce cardiomyocytes apoptosis. The main way of cardiomyocyte death is apoptosis during reperfusion processes.

参考文献/References

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备注/Memo

备注/Memo:
收稿日期:2008-01-22.基金项目:国家自然科学基金课题资助(30570758,30770847);陕西省科学技术研究发展计划项目资助(2005K13G14) 通讯作者:王晓明,副教授,博士,主要从事心血管疾病的基础与临床研究Email:xmwang@fmmu.edu.cn 作者简介:曹亚南,医师,硕士生Email:cynthiaa@fmmu.edu.cn
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