【作者】 李鸿珠； 徐长庆； 韩丽萍； 姜春明； 李弘； 郭津； 李光伟； 高君；

【Author】 Hongzhu Li Changqing Xu Liping Han Chun-ming Jiang Hong LI Jin Guo Guangwei L Jun Gao Department of Pathophysiology,Department of Biochemistry,Harbin Medical University;The Fourth Orthopaedics Department of The First Clinical College of Harbin Medical University,Harbin,China,150086

【机构】 哈尔滨医科大学病理生理教研室； 黑龙江生物医药工程重点实验室； 哈尔滨医科大学第一临床医院骨四科；

【摘要】 目的:观察大鼠心肌组织多巴胺受体(DR)在缺血/再灌注不同时间的表达规律及DR激活对心肌细胞凋亡的影响。方法:采用Lagendorff离体心脏灌流的方法复制心脏缺血/再灌注(I/R)模型;乳鼠心肌细胞原代培养复制缺氧/复氧模型。RT-PCR检测DR1、DR2 mRNA的表达:Western blotting检测DR1、DR2、Bcl-2、caspase-3,-8,-9、cytochrome c(Cyt c)、Fas/Fas-L蛋白质的表达;激光共聚焦显微镜检测细胞内钙浓度;紫外分光光度计测定大鼠冠脉流出液和培养液中LDH、SOD、MDA的水平;流式细胞仪、TUNEL染色、MTT检测心肌细胞的凋亡率和存活率;透射电镜观察心肌超微结构的变化。结果:(1)大鼠心肌组织DR1、DR2 mRNA和蛋白表达在缺血1h时高于正常对照组(P<0.05),缺血1h后再灌1h显著高于正常对照组(P<0.01),2h达高峰,3h、4h后降低,但仍高于正常组(P<0.01);同时随再灌时间延长,冠脉流出液中LDH活性增加,心肌超微结构损伤加重;(2)与I/R组比较,SKF-38393(DR1激动剂)组LDH活性增加,SOD活性降低,MDA含量增加,caspase-3,-8,-9、Fas/Fas-L、cytochrome c(Cyt c)蛋白表达增加(P<0.05,P<0.01),Bcl-2蛋白的表达降低(P<0.05),细胞凋亡率升高(P<0.05),细胞存活率降低(P<0.05),细胞内钙浓度升高(P<0.05),心肌细胞损伤加重;Bromocriptine(DR2激动剂)组对上述指标的影响与SKF-38393相反;SCH-23390(DR1抑制剂)、Haloperidol(DR2抑制剂)对上述指标的影响不明显。结论:(1)大鼠心肌多巴胺受体DR1、DR2表达在缺血/再灌注损伤过程中呈先升高后降低的特点,可能参与心肌缺血,再灌注损伤的发生;(2)DR1激活可促进心肌细胞凋亡,DR2则抑制心肌细胞凋亡,其信号传导通路均与Cyt c-caspase-3和Fas/Fas-L途径有关。更多还原

【Abstract】 Objective;To investigate the expression of dopamine receptor （DR） in rat cardiac tissue during ischemia/reperfusion injury （IR1）,and the effect of DR activation on cardiomyocytes apoptosis. Methods;The rat cardiac IRl model in vitro was done using Langendorf device;rat cardiomyocytes IRi model of primary cultured neonatal was created by anoxia-reoxygenation.RT-PCR was used to detect the mRNA expression of DRland DR2;the protein expressions of DRI,DR2,Bcl-2,caspase-3,-8,-9, cytochrome c （Cyt c）,Fas,Fas-L were observed using Western blot;the intracellular free-calcium concentration ([Ca²⁺]_i) was detected through Laser Confocal Scanning Microscope;the level of LDH,SOD and MDA in the coronary effluent and cultural medium was assayed with ultraviolet spectrophotometer;the survival and apoptotic rate of cardiomyocytes were examined by MTT,TUNEL staining and flow cytometric;the ultrastructure of cardiac tissue was determined with transmission electron microscope. Results;（1） The mRNA and protein expression of DR1 and DR2 in rat cardiac tissue were increased at ischemia lh （P<0.05）,marked increased at reperfusion 1h （P<0.01）,reached the highest level at 2h,but lightly decreased at 3h and 4h;the activity of LDH was increased and the myocardial ultrastructure was injured seriously after reperfusion;（2） Compared with I/R group,in SKF-38393 （DRI agonist） group,the LDH activity was obviously increased,the SOD activity was decreased,the MDA contents were increased, the expressions of caspase-3,-8,-9,Fas/Fas-L,Cyt c proteins were up-regulated （P<0.05,P<0.01）,the expression of Bcl-2 protein was down-regulated （P<0.05）,and the apoptotic rates of cardiomyocytes were increased,and the cardiomyocyte viability was reduced and [Ca²⁺]_i was increased,and cardiomyocytes injury was aggravated.The effect of Bromocriptine （DR2 agonist） on the above -mentioned index was opposite to SKF-38393;SCH-23390 （DR1 antagonist） and Haloperidol （DR2 antagonist） had no significant effect on IRI. Conclusions;（1） The expression of DR1,DR2 in rat cardiac tissue is firstly increases,and then decreases during I/R,and both DR1 and DR2.are involved in the cardiac ischemia-reperfusion injury;（2） DR1 activation promotes apoptosis,DR2 activation inhibits apoptosis, which signal transduction mainly through Cyt c-caspase-3 and Fas/Fas-L pathways.更多还原