【作者】 李欣；

【导师】 赵春燕；

【作者基本信息】 吉林大学 ， 生理学， 2007， 博士

【摘要】 溶血磷脂酸(lysophosphatidic acid ,LPA)是细胞膜磷脂代谢的中间产物。急性心肌梗死时由于血小板的聚集可产生并释放大量的LPA,而心肌细胞上又存在着对LPA敏感的特异性G-蛋白偶联受体。论文对LPA血浆浓度显著升高的卵巢癌患者和其它腹腔恶性肿瘤患者的心律失常发生率进行了比较,发现卵巢癌患者心律失常发生率明显高于其它腹腔恶性肿瘤患者的心律失常发生率;以体表心电为指标观察到心梗模型大鼠在给予外源性LPA后心律失常发生率增加;利用LPA试剂盒测定大鼠血浆LPA浓度,观察到心梗发生心律失常组的血浆LPA浓度明显升高;利用离体心脏灌流法观察到外源性给予LPA后离体心脏的心律失常发生率增加;在考察LPA对豚鼠乳头肌动作电位参数的影响时,发现LPA可显著延长乳头肌动作电位时程,增加动作电位幅值,并使心肌收缩力增强;利用膜片钳技术观察到LPA显著抑制心肌细胞延迟整流钾通道和内向整流钾通道;利用激光扫描共聚焦显微系统观察到LPA可增加心肌细胞游离钙浓度。上述结果提示:(1)LPA显著抑制心肌细胞延迟整流钾电流和内向整流钾电流,使细胞膜电位降低,易形成单向阻滞和慢传导,从而形成折返激动,可引起折返性心律失常,并且易诱发尖端扭转性室速或频发的单型性室性心律失常;而膜电位的降低使阈值降低,提高了心肌细胞的兴奋性,易引起异位心律;同时膜电位降低易引发早期后除极(EADs),产生触发激动。(2)LPA使心室肌细胞钙浓度升高,产生钙超载导致后除极(DADs),引发心律失常;(3)LPA可增加心肌细胞动作电位幅值(APA),主要由Na+电流经细胞膜钠通道所引起。钠电流的增加使心肌细胞不应期缩短,传导速度加快,易引起折返激动;同样会使原本没有自律性的细胞产生自律性,形成异位心律。本文从整体、器官、细胞分子多个水平,利用心梗模型、LPA试剂盒测定LPA浓度、离体心脏灌流术、乳头肌动作电位参数测定、膜片钳技术测定膜电流、激光扫描共聚焦显微系统测定细胞内钙浓度等多种技术系统的研究了LPA在急性心肌梗死诱发心律失常中的作用及机制。本文的创新之处:(1)观察到大鼠心肌梗死组血浆LPA浓度明显高于对照组,心肌梗死并发生心律失常组大鼠的血浆LPA浓度高于单纯心梗组;(2)观察到LPA可显著延长豚鼠乳头肌动作电位时程,增加动作电位幅值,并使心肌收缩力增强;(3)观察到LPA可显著抑制豚鼠心室肌细胞延迟整流钾通道,并呈剂量依赖性。LPA抑制心肌细胞延迟整流钾通道的作用可被Gi蛋白阻断剂PTX所阻断;(4)观察到LPA可抑制豚鼠心室肌细胞内向整流钾通道,并可被Gi蛋白阻断剂PTX所阻断;(5)观察到LPA可增加大鼠乳鼠培养心肌细胞的游离钙浓度,并可被LPA特异性受体阻断剂Ki16425所阻断。更多还原

【Abstract】 The investigation about the global death cause in 1990 by World Health Organization indicated that the ischemic heart disease is on the top. Acute myocardial infarction (AMI) have become to be important disease hazarding human health. The ratio of AMI patients companioning with arrhythmia is 75%-95%. Especially in the ten days of AMI onset , the incidence of all kinds of arrhythmia could become 86%-100%. Arrhythmia is the important death cause in AMI.Lysophosphatidic acid is an intermediary metabolite of phospholipid in cell membrane. Recent studies have shown that LPA exhibits a wide spectrum of biological effects on variety cells via G-protein-coupled endothelial-cell differentiation genes(EDG) receptors. Clinical study indicated that the concentration of LPA could increase compared with control group in the ischemic heart disease, such as acute myocardiac infarction and angor pectoris. The reason lie in that (1) phospholipase are activated in AMI. The ability of cleavage makes the product enrich. (2)The important source of LPA comes from activated platelets. Onset of these diseases could accumulate more platelets. So the concentration of LPA increases immediately. LPA receptors are distributed in myocytes. All these suggest that LPA may play an important role in arrhythmia occurrence complicated by acute myocardial infarction. The relationship between LPA and arrhythmia complicated by acute myocardial infarction have not been reported expect our lab. In order to find the relationship between LPA and arrhythmia complicated by acute myocardial infarction, we investigated the effect of LPA in organ、tissue、cell and molecule different level. We expected to obtain a theoretical explanation and supplement of the mechanism of arrhythmia complicated by acute myocardial infarction. Moreover, we also excepted to find new target point link of LPA for prevention and therapy of this kind of arrhythmia.Ⅰ. The epidemiology investigation about incidence of arrhythmia in ovary cancer and hepatoma.The study indicated the relationship between LPA and arrhythmia is close.The plasma LPA concentration of ovary cancer patients is obviously higher. We made a clinic epidemiology investigation about incidence of arrhythmia in ovary cancer and hepatoma. We observed that the incidence of arrhythmia of ovary cancer is higher than hepatoma group (p<0.05).This result suggests that LPA may involve in arrhythmia occurance.Ⅱ. The effect of LPA on the incidence of arrhythmia in acute myocardial infarctional rat.ECG of rats was used as an index of present study. After increasing the concentration of LPA in the local region of myocardial infarction, the times of extrasystole occurrence were obviously higher than those of model control group (p <0.05). But if pretreated with the antagonist of G protein PTX before administrated with LPA, there was no significant difference compared with model control group (p>0.05). All these suggested that LPA could induce the arrhythmia occurrence after acute myocardial infarction via a G-protein-coupled receptors pathway.Ⅲ.The plasm concentration of LPA in acute myocardial infarctional rat. LPA test box was used to record the plasm concentration of LPA in acute myocardial infarctional rat. We observed that the concentration of LPA in the AMI group were obviously higher than those of model control group. the concentration of LPA in the AMI accompand with arrhythmia group were higher than AMI group(p<0.05).It further suggests that LPA may involve in arrhythmia occurance.Ⅳ. The experiment of arrthimia induced by LPA in isolated rat hearts.In order to eliminate the factor of nerve the Langendorff perfusion method was used. After administration with LPA in perfused hearts, the times of extrasystole occurrence were higher than those of model control group But if pretreated with the antagonist of G protein PTX 0.1 mg·L-1before administrated with LPA, there was no significant difference compared with model control group (p>0.05). All these suggested that LPA could induce the arrhythmia occurrence after acute myocardial infarction via a G-protein-coupled receptors pathway.Ⅴ. The effect of LPA on action potential and contraction force in guinea pigs papillary muscles.To investigate the effect of LPA on action potential, standard microelectrodes and force transducer were used. LPA could increase contraction force, the action potential amplitude(APA), prolong the 50% repolarization levels and the 90% repolarization levels in guinea pigs in a dose-dependence manner compared with themselves before administration. TEA shortened the APD compared with LPA 10μmol·L-1 group(P<0.05).These indicate that LPA could increase action potential amplitude, prolong action potential duration and increase contraction force and the effect of LPA on APD could exhibit via inhibiting potassium pathway.Ⅵ. Effects of LPAon the potassium current of ventricular myocytes in guinea-pig.The whole cell patch clamp technique was used to record the potassium current of ventricular myocytes, which was stimulated from holding potential -50 mV stepping from -100 to +50 mV to elicit“N”shape potassium current curve. The result suggested that LPA could decrease delayed rectifier potassium current and inward rectifier potassium current in a dose-dependence manner.Ⅶ. Effects of LPA on the delayed rectifier potassium current and inward rectifier potassium currentThe whole cell patch clamp technique was used to record the delayed rectifier potassium current of ventricular myocytes, which was from holding potential -50 mVstepping from -50 to +50 mV. The whole cell patch clamp technique was used to record the inward rectifier potassium current of ventricular myocytes, which was from holding potential -40 mV stepping from -100 to +30 mV. The results suggested that LPA could obviously decrease delayed rectifier potassium current and inward rectifier potassium current in a dose-dependence manner. The effects of LPA decreasing the Ik and Ik1 were significantly blocked by PTX, which suggested that the inhibiting effect of LPA on Ik and Ik1channel may be modulated by G protein pathway.Ⅷ. Measurement of intracellular free calcium concentration of ventricular myocytes by using furo-3-AM fluorescence dye.Supervise the dynamic change of intracellular free calcium concentration of ventricular myocytes under LSCM continuous scanning. The result was that after application LPA 10μmol·L-1, the intracellular free calcium concentration was increased significantly. Moreover, pretreated with the antagonist of LPA receptors Ki16425 before administrated with LPA, there was no significant difference compared with model control group (p>0.05). This result was consistent with the above contraction force experiment result. These results indicate LPA could increase the intracellular free calcium concentration of ventricular myocytes via LPA receptors.All the results above suggest that LPA may be involved in the process of arrhythmia occurrence complicated by acute myocardial infarction. Its mechanism may be: LPA can prolong the action potential duration and decrease the membrane potential by inhibiting delayed rectifier potassium current and inward rectifier potassium current in a dose-dependence manner. These effects can increase the auto-rhythmicity of cardiac myocytes and induce re-entry activity as well as ectopic rhythm; LPA can increase the intracellular calcium concentration and induce intracellular calcium overload which tend to induce the generation of EADs and DADs.更多还原