【作者】 樊磊；

【导师】 梅其炳； 张峰；

【作者基本信息】 第四军医大学 ， 药理学， 2010， 硕士

【摘要】 三磷酸腺苷(ATP)作为细胞外信号分子的功能早已受到人们的关注并得到研究确认,很多类型的细胞在一定条件下均可释放ATP等核苷酸分子；ATP还是神经末梢递质,参与神经兴奋传导。由于核苷酸类受体的多样性以及物种间的差异,胞外核苷酸及其受体的生理功能还不是非常明确。胞外尿苷三磷酸(UTP)与ATP同是信号递质,参与机体生理病理过程的调节。P2受体是嘌呤与嘧啶受体(P receptors, receptors for purines and pyrimidines)的一大类,其内源性配体主要是ATP和UTP以及它们的水解产物。P2受体分为配体门控离子通道受体——P2X受体和G-蛋白偶联离子通道受体——P2Y受体,这两类受体又有诸多亚型。这些受体亚型虽然结构相似但生理功能及药理性质有很大差异,并且各种亚型之间对于同种配体的亲和力不同。与ATP相比,对于UTP也是一种细胞外信号分子的认识较晚,细胞可自分泌或者旁分泌UTP的假说最近才得到实验支持,UTP的研究成为了新的热点。细胞持续释放UTP以维持正常的生理功能,在一些病理情况下细胞也可释放UTP,例如心肌梗塞后冠脉窦血液中的UTP浓度升高。研究发现,UTP预处理的心肌细胞对缺氧/复氧损伤的耐受性提高,在体实验表明UTP可改善大鼠心肌梗塞后的心脏功能和形态学指标。UTP预处理保护心肌的作用机制还不明确,而且,尿苷的类似物腺苷可通过触发/介导缺血预处理效应而保护心肌。本研究旨在明确介导UTP心肌预保护效应的受体以及阐明其可能的机制。本研究分为两部分：一、间接体内实验：即活体给药后的离体心脏灌流实验实验目的：探讨P2Y受体激动剂UTP对于大鼠心脏缺血/再灌注损伤(I/RI)的延迟性保护作用。实验方法：将24只SD(Sprague—Dawley)大鼠随机分为4组：对照组(静脉注射9g/L生理盐水)、UTP组(静脉注射UTP 4.4μg/kg)、UTP+苏拉明(suramin,SRM)组(静脉注射4.4μg/kg UTP前5 min注射30μg/kgSRM)及SRM组(静脉注射30μg/kg SRM)。所有大鼠尾静脉给药24 h后,建立Langendorff离体心脏灌流模型。平衡10 min后全心停灌,25 min后复灌,持续再灌注40min。记录心脏缺血/再灌注前后血流动力学指标,观察心肌超微结构,记录心脏表面心电图计算心律失常的发生频率,收集冠脉流出液,用全自动生化分析仪测量乳酸脱氢酶(LDH)的释放量。将剩余心肌组织用4%多聚甲醛溶液固定后包埋切片,免疫荧光染色确认成熟大鼠心脏中是否存在P2Y2受体和P2Y4受体。实验结果：复灌后心脏复跳的第5 min和25 min时,UTP组的左室发展压(LVDP)、等容收缩期左心室内压力上升/下降最大速率(±dp/dtmax)恢复率均优于对照组(P<0.01)；冠脉流出液中LDH的水平明显值降低(P<0.01)；复灌第5-15 min和第25-35min时的心律失常的发生频率均显著下降(P<0.01)；心肌超微结构的损伤减轻。而以SRM与UTP同时作用后,UTP对心脏的保护作用则被取消。SRM组与对照组相比各项指标无明显变化。心肌组织切片的免疫荧光染色表明,成熟大鼠的心肌确实分布有P2Y2受体和P2Y4受体。结论：UTP预处理可对心脏I/RI产生延迟性拮抗作用；而P2Y受体拮抗剂SRM可取消这种作用,表明UTP对心脏的保护作用是通过P2Y受体介导的,由于UTP对P2Y2受体和P2Y4受体亲和力较高且这两种受体广泛分布于成熟大鼠的心肌,那么UTP的心肌保护作用可能是通过这两个P2Y受体的亚型介导的。二、心肌细胞实验实验目的和方法1、ATP对心肌细胞缺氧/复氧损伤的影响目的：通过观察ATP预处理心肌细胞是否和UTP一样具有拮抗缺氧/复氧损伤的作用探讨介导UTP类物质心肌保护作用的P2Y受体亚型。方法：实验分组：①正常对照组；②阳性对照组即缺氧/复氧组(缺氧12h,复氧4h)；③低剂量ATP预处理组(缺氧/复氧处理前24h加3μM ATP);④中剂量ATP处理组(ATP15μM)；⑤高剂量ATP(ATP 50μM)。用全自动生化分析仪检测细胞复氧液中LDH的释放量。2、UTP对心肌细胞缺氧/复氧后细胞凋亡的影响目的：通过流式细胞仪检测UTP对于心肌细胞缺氧/复氧损伤引起凋亡的抑制作用。方法：实验分组：①正常对照组；②阳性对照组即缺氧/复氧组(缺氧12h,复氧4h)③UTP预处理组(缺氧/复氧处理前24h加50μM UTP);④UTP和SRM处理组(SRM 300μM,15min后加UTP50μM)。收集缺氧/复氧处理后的心肌细胞,进行流式细胞仪检测。3、Ca2+在UTP保护心肌缺氧/复氧损伤中的作用目的：明确细胞外Ca2+在P2Y受体介导的UTP心肌预保护中的作用。方法：实验分组：①正常对照组；②阳性对照组即缺氧/复氧组(缺氧12h,复氧4h)；③含Ca2+培养基UTP预处理组(缺氧/复氧处理前24h加UTP 50μM)；④无Ca2+培养基UTP处理组(缺氧/复氧处理前24h用无钙培养基置换原有培养基,加UTP50μM,再加入含钙培养基)。用全自动生化分析仪检测细胞复氧液中LDH的释放量。实验结果：1、ATP预处理的心肌细胞缺氧/复氧后,与对照组相比,低剂量组和中剂量组细胞培养液中LDH的释放量没有差别,高剂量组的LDH释放量增多。2、UTP预处理的心肌细胞凋亡早期百分率较对照组明显降低,而SRM与UTP合用时,凋亡早期的心肌细胞百分率升高。3、与对照组相比,无Ca2+培养基和含Ca2+培养基中的心肌细胞在缺氧/复氧处理后LDH的释放量均减少。而无Ca2+培养基组和含Ca2+培养基组之间LDH的释放量没有显著性差异。结论：1、ATP预处理不能产生心肌预保护作用,由于大鼠P2Y4受体与UTP和ATP的亲和力相当而P2Y2受体对ATP的亲和力较UTP差,那么可推断UTP的心肌保护作用主要是由P2Y2受体介导的。2、UTP预处理可通过抑制细胞的凋亡而保护心肌细胞。3、UTP作用于无Ca2+培养基中的心肌细胞后细胞抗缺氧/复氧损伤的能力并不降低说明该效应不依赖胞外Ca2+的内流。更多还原

【Abstract】 The physiological significance of ATP as extracellular signalling molecules is well established. ATP is released in a regulated manner from many types of cells,and released nucleotides act as the transmitters from the nerve terminal. Because of the species difference and the diversity of receptors, the physiological functions of extracellular nucleotides were less well understood. Like ATP, as a signal transmitter, UTP regulates physiological and pathological processes of the body. P2 receptor which is a category of P receptor.,Its endogenous ligands are ATP and UTP, and their hydrolysis products—ADP and UDP. P2 receptor is divided into ligand-gated ion channel receptor-P2X and G-protein-coupled receptor-P2Y. These two types of receptors have many subtypes.In contrast to the compelling evidence for the extracellur signaling role of ATP,the hypothesis that UTP may also fufill an autocrine/paracrine role has only recently gained experimental support. The research of UTP become a new hot spot. Under normal conditions, cells continue to release UTP in order to maintain the normal physiological function. In some pathological cases, cells also can release UTP. For example, after myocardial infarction, the concentration of UTP increased in the coronary sinus blood. The source of UTP may be myocardial cells and vascular endothelial cells. In other studies,they found that myocardial tolerance to hypoxia/reoxygenation injury enhanced when pretreatment with UTP.In vivo experiments also showed that UTP can improve cardiac function and morphological index after myocardial infarction in rats. However the mechanism of UTP preconditioning is not clear, moreover, adenosine which is the analogues of uridine can mediate the effects of myocardial ischemic preconditioning. This study was designed to discover the receptor which specifically mediate the preconditioning protective effect of UTP as well as to clarify its possible mechanism.This study was divided into two partsPart 1.Indirect in vivo experiment:isolated heart perfusion experiment after administration of drugs in vivo.Aim:To explore the antagonistic effect of uridine triphosphate (UTP) on myocardial ischemia/reperfusion injury(I/RI) in rats.Methods:Twenty four SD rats were randomly divided into 4 groups: control group (9 g/L normal saline, i.v.), UTP group(4.4μg/kg i.v.), UTP and suramin(SRM, antagonist of P2Y receptor) group (4.4μg/kg i.v.+30μg/kg i.v.) and SRM group(30μg/kg i.v.).24 h after drug injection, hearts were isolated from the rats and perfused with K-H solution by Langendorff system. Hearts were subjected to 25 min ischemia followed by 40 min reperfusion. The hemodynamics indexs were measured and ECG was recorded. The coronary effluent was collected for measuring lactate dehydrogenas(LDH) level. Ultrastructure of myocardial was observed by transmission electron microscope. The remaining myocardial tissue was fixed with 4% paraformaldehyde and embedded for slice, samples of myocardial tissue were prepared for immunofluorescence staining to confirme the existence of P2Y2 receptors and P2Y4 receptors in rats’hearts.Results:After 25 min ischemia, the UTP group had significantly better recovery percentage of left ventricular function (P<0.01) and lower LDH level(P<0.01) compared with control group, the arhythmia frequency was also lower(P<0.01) than control group. The myocardial ultrastructure of UTP group was generally normal. The cardio-protective effect of UTP were eliminated by SRM. Myocardial biopsy and immunofluorescence staining showed that the distribution of P2Y2 receptor and P2Y4 receptors in mature rat heart.Conclusion:UTP pretreatment can protect rat’s hearts from I/RI, which is cancelled by the P2Y receptors’antagonist suramin, indicating that the myocardial protective effect of UTP is mediated by P2Y receptors. As the affinity of UTP to the receptors of P2Y2 and P2Y4 is high and these two receptors are widely distributed in the mature myocardium, then the myocardial protective effect of UTP may be mediated through these two subtypes of P2Y receptor.Part2. Myocardial cells experimentsAim and Methods: 1.ATP on hypoxia/reoxygenation injury.Aim:To investigate whether ATP preconditioning can protect myocardial cells from hypoxia/reoxygenation injury like UTP.Methods:Experimental groups:①normal control group;②positive control group which is hypoxia/reoxygenation group (hypoxia 12h, reoxygenation 4h);③low dose of ATP pretreatment group (pretreatment of 3μM ATP 24h prior hypoxia/reoxygenation);④middle dose of ATP treatment group (ATP15μM);⑤high dose of ATP treatment group (ATP 50μM).2. Effect of UTP on apoptosis of myocardial cell after hypoxia/ reoxygenation injury.Aim:To investigate the inhinbition effect of UTP to cardiomyocyte apoptosis which induced by hypoxia/reoxygenation injury.Methods:Experimental groups:①normal control group;②positive control group which is hypoxia/reoxygenation group (hypoxia 12h, reoxygenation 4h)③UTP preconditioning group (pretreatment of 50μM UTP 24h prior hypoxia/ reoxygenation);④UTP and SRM handling group (SRM 300μM,15min later treatment with 50μMUTP). Collection of hypoxia/reoxygenation treated myocardial cells were detected by flow cytometry.3. The effect of Ca2+in UTP preconditioning before myocardial hypoxia/ reoxygenation injuryAim:To investigate the role of extracellular Ca2+in P2Y receptor-mediated cardio-protective effect of UTP preconditioning.Methods:Experimental groups:①normal control group;②positive control group which is hypoxia/reoxygenation group (hypoxia 12h, reoxygenation 4h);③The Ca2+ containing medium UTP preconditioning group (24h before hypoxia/reoxygenation treatment with 50μM UTP);④The Ca2+-free medium UTP preconditioning group (Before hypoxia/reoxygenation treatment, the original medium was replaced by Ca2+-free medium and treatment with 50μMUTP, then replaced with Ca2+containing medium). The LDH released from the myocardial cells after hypoxia/reoxygenation treatment was analyzed using Automatic biochemical analyzer.Results:1. The release of LDH from the myocardial cells which were pretreated with low dose and mid dose of ATP after hypoxia/reoxygenation showed no significant difference compared with the control group,.The release of LDH in the high dose group increased compared with the control group.2. The percentage of early apoptosis in UTP pretreatment group was markedly decreased, while SRM combined with UTP pretreatment can increase the percentage of early apoptosis in myocardial cells.3. In compared with the control group, the release of LDH after hypoxia/ reoxygenation in the Ca2+-free medium and medium containing Ca2+reduced significantly. However, difference was found little between these two groups.Conclusions:1. ATP preconditioning can not produce protective effect on myocardial cells. As the affinity of rat’s P2Y4 receptor to UTP is the same with ATP, but ATP could not active P2Y2 receptor fully, so we may conclude that myocardial protection of UTP is mainly mediated by the receptor of P2Y2 rather than P2Y4 receptor.2. Pretreatment of UTP can inhibit apoptosis of myocardial cells,this may be the reasen of UTP induced myocardial-protection. 3. When UTP acting on myocardial cells in the Ca2+-free medium, the ability of anti-hypoxia/reoxygenation injury did not compromised. It may elucidate that the mechanism of myocardial-protection is not related with the extracellular Ca2+.更多还原