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多巴胺受体对大鼠心肌缺血/再灌注损伤的影响

Effect of dopamine receptor on myocardial ischemia/reperfusion injury in rat

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【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。

【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.

【基金】 国家自然科学基金资助项目(No.30370577,30470688);黑龙江省教育厅海外学人科研(合作)资助项目(N0.1053HQ010)。
  • 【会议录名称】 中国病理生理学会受体和信号转导专业委员会暨消化专业委员会联合学术会议论文汇编
  • 【会议名称】中国病理生理学会受体和信号转导专业委员会暨消化专业委员会联合学术会议
  • 【会议时间】2008-11
  • 【会议地点】中国广东广州
  • 【分类号】R363
  • 【主办单位】中国病理生理学会
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