Suzuki K, Sawa Y, Kaneda Y, Ichikawa H, Shirakura R, Matsuda H
First Department of Surgery, Osaka University, Japan.
J Mol Cell Cardiol. 1998 Jun;30(6):1129-36. doi: 10.1006/jmcc.1998.0678.
Previous studies indicate that heat shock protein 70 (hsp70) improves the myocardial tolerance to ischemia-reperfusion injury by a mechanism that is not well understood. To better define this protective function, it is important to distinguish a role of hsp70 on coronary endothelial cells (cEC) from that on cardiac myocytes. Thus, we transfected rat cEC with a human hsp70 cDNA by using hemagglutinating virus of Japan-liposome method (group H). Control cells (group C) were transfected with a vector containing no gene. Immunohistochemical staining demonstrated overexpression of hsp70 in the cytosol of the cells in group H. Western blotting also showed large amounts of hsp70 expression in these cells. After 18 h of hypoxia followed by 2 h of reoxygenation, the adenosine triphosphate content was higher in group H (H v C; 1.05 +/- 0.08 v 0.68 +/- 0.04 microgram/dish, P = 0.0007). In addition, lactate dehydrogenase leakage after hypoxic insult was lower in group H than that in group C (61.3 +/- 4.5 v 85.4 +/- 6.1 10(-3) IU/dish/37 degrees C, P = 0.004). Conversely, the leakage of FITC-albumin through a confluent monolayer of cEC after hypoxia-reoxygenation was less in group H than that in group C (11.1 +/- 1.8 v 27.4 +/- 3.1%, P = 0.0003). Thus, the high level expression of hsp70 caused by gene transfection enhanced the hypoxic tolerance of coronary endothelial cell. Therefore, coronary endothelial cell is an important targets of hsp70-mediated cardioprotection as well as cardiac myocytes.
先前的研究表明，热休克蛋白70（hsp70）可通过一种尚未完全明确的机制提高心肌对缺血-再灌注损伤的耐受性。为了更好地界定这种保护功能，区分hsp70在冠状动脉内皮细胞（cEC）和心肌细胞上的作用很重要。因此，我们采用日本血凝病毒-脂质体法将人hsp70 cDNA转染至大鼠cEC（H组）。对照细胞（C组）用不含基因的载体转染。免疫组织化学染色显示H组细胞胞质中hsp70过表达。蛋白质印迹法也显示这些细胞中有大量hsp70表达。缺氧18小时后再复氧2小时，H组的三磷酸腺苷含量更高（H组对C组；1.05±0.08对0.68±0.04微克/培养皿，P = 0.0007）。此外，H组缺氧损伤后的乳酸脱氢酶漏出量低于C组（61.3±4.5对85.4±6.1×10⁻³国际单位/培养皿/37℃，P = 0.004）。相反，缺氧-复氧后，H组中通过融合的cEC单层的异硫氰酸荧光素-白蛋白漏出量少于C组（11.1±1.8对27.4±3.1%，P = 0.0003）。因此，基因转染导致的hsp70高水平表达增强了冠状动脉内皮细胞的缺氧耐受性。所以，冠状动脉内皮细胞与心肌细胞一样，是hsp70介导的心脏保护作用的重要靶点。
