中文摘要：

目的观察腺病毒介导的鞘氨醇激酶1（SPK1）高表达对心肌缺血再灌注损伤的保护作用。方法Wistar大鼠心肌内注射携带人SPK1基因的复制缺陷型重组腺病毒（Ad-SPK1）,以5×109pfu/ml的病毒总量分4点注射,对照组心肌内注射同等量的携带绿色荧光蛋白基因的重组腺病毒（Ad-GFP）,3d后进行离体心脏缺血再灌注实验,测定左室舒张末压（LVEDP）、左室收缩压（LVSP）,观察心律失常的发生情况,测定肌酸激酶（CK）的释放水平,用酶学方法检测SPK1的活性,用薄层层析法及Western blot方法检测外源和内源性SPK1在心肌组织的表达。结果心肌内注射Ad-SPK1的大鼠3d后心肌组织SPK1激酶活性明显升高,比对照组升高5倍左右;与对照组相比,注射Ad-SPK1的大鼠心脏对缺血再灌注损伤有明显的抵抗能力,表现在心脏的收缩和舒张功能在缺血和再灌注过程中没有受到明显损伤,心律失常的发生明显减少以及再灌注过程中CK释放明显减少。结论腺病毒介导的SPK1基因转染能预防缺血再灌注导致的心脏损伤。

英文摘要：

Objective Sphingosine kinase 1 （SPK1） has been identified as a central mediator of ischemia preconditioning, and it has been shown to protect reactive oxygen species （ROS）-induced cardiomyocytes death. The present study aims at investigating the protective effects of adenovirus-mediated sphingosine kinase 1 gene （Ad-SPK1） transfer on ischemia-reperfusion induced cardiac injury. Methods Wistar rats were anesthetized and a left thoracotomy was performed. About 5×108 PFU of Ad-SPK1 in total volume of 100μl was injected intramyocardially into four separate sites of the left ventricular wall with a 30-gauge needle. The control rats received the same injection of adenovirus carrying green fluorescent protein gene （Ad-GFP）. Three days later, hearts were isolated and subjected to ischemia/reperfusion （I/R） （30min/30min） ex vivo. Heart performance was evaluated by measuring the left ventricular systolic pressure （LVSP） and left ventricular end-diastolic pressure （LVEDP）. The incidence of arrhythmia was recorded. Cardiomyocyte viability was detected by the detection of the release of creatine kinase （CK）. The cardiac SPK1 activity was measured using an enzyme method. The forced and the total SKP1 expression was analyzed by immunoblotting with anti-FLAG and anti-SPK1 antibodies. Results The cardiac SPK1 activity was increased by about five-fold by injection of Ad-SPK1, compared to Ad-GFP control group. A more potent performance and a lower incidence of arrhythmia were observed in Ad-SPK1-injected hearts during the reperfusion period, compared with Ad-GFP-injected ones. Enzymatic activity assay showed that creatine kinase release was also less in Ad-SPK1-injected hearts. Conclusion Adenovirus-mediated SPK1 gene transfer efficiently protects heart from injury induced by ischemia-reperfusion.