Han Liping, Xu Changqing, Jiang Chunming, Li Hongzhu, Zhang Weihua, Zhao Yajun, Zhang Li, Zhang Yanqiao, Zhao Weiming, Yang Baofeng
Department of Pathophysiology, Harbin Medical University, Harbin 150086, China.
Cell Biol Int. 2007 Nov;31(11):1345-52. doi: 10.1016/j.cellbi.2007.05.015. Epub 2007 May 29.
We incubated neonatal Sprague-Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 micromol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2+]i. However, pretreatment with 10 micromol/l putrescine had the opposite effects. Evidence for this "double-edged" effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.
我们将原代培养的新生Sprague-Dawley大鼠心肌细胞置于模拟缺血的培养基（由缺氧加血清剥夺组成）中孵育2小时，然后在正常培养基中再孵育24小时，以建立模拟缺血/再灌注（I/R）损伤模型。通过MTT法、TUNEL染色和流式细胞术检测凋亡细胞死亡。通过透射电子显微镜评估形态学改变，通过蛋白质印迹法检测半胱天冬酶-3和-9、Bcl-2的表达以及细胞色素c的释放，并通过激光共聚焦扫描显微镜检测细胞内游离钙浓度（[Ca2+]i）。结果表明，用10微摩尔/升精胺或亚精胺预处理可显著抑制I/R细胞中的凋亡，抑制半胱天冬酶-3和-9的表达以及细胞色素c的释放，上调Bcl-2表达并降低[Ca2+]i。然而，用10微摩尔/升腐胺预处理则产生相反的效果。首次提出了多胺对I/R损伤心肌细胞凋亡的这种“双刃剑”作用的证据。这可能提示了预防和治疗心脏缺血/再灌注损伤的新药理学靶点。
