【作者】 吉恩生；

【导师】 何瑞荣；

【作者基本信息】 河北医科大学 ， 生理学， 2004， 博士

【摘要】 植物性雌激素是主要来源于豆类植物，是一类天然存在于植物中的具有双酚环结构的生物活性物质，其结构和功能与雌激素近似，它可与雌激素受体结合，具有雌激素样心血管效应。植物性雌激素主要包括异黄酮、木酚素和香豆素三类。三羟异黄酮（genistein, GST）属于植物性雌激素中的异黄酮类。已有大量研究表明，以GST 为代表的植物性雌激素对于心血管系统具有良好的保护作用。例如，GST具有较强的抗动脉粥样硬化和舒血管效应，抑制细胞增殖和新生血管的形成，从而对心血管有保护和调节作用。先前的实验表明，GST可可剂量依赖性地缩短正常乳头肌动作电位时程（APD）；对部分去极化乳头肌，GST除缩短动作电位时程外，还使动作电位幅值(APA)和超射值(OS)降低，零相最大上升速度减慢(Vmax)；预先给予GST可剂量依赖性地抑制哇巴因所引起的豚鼠乳头肌的迟后去极化（DAD）及触发活动（TA）；GST抑制窦房结和房室结起搏细胞的0相上升最大速度，舒张期除极速度(VDD)，起搏放电频率(RPF)和动作电位幅度；GST还抑制人的心房肌纤维的舒张期除极速度和起搏细胞放电频率(RPF)；认为上述效应是由于GST通过非NO途径抑制Ca2+内流所致。然而，有关GST其他的心血管效应及其机制，仍不明确。本实验通过对在体和离体的动物实验，在下列几方面进行了研究：（1）采用离体血管环灌流方法，观察GST对家兔股动脉张力的影响，并探讨其影响机制。（2）采用后肢、肾脏和肠系膜动脉在体恒流灌注法，观察向灌流环路中直接注射GST的血管效应。（3）采用家兔心脏缺血/再灌注损伤模型，观察GST对家兔心脏缺血/再灌注损伤模型的影响。（4）用Fluo3-AM负载分离的豚鼠心室肌细胞后，用激光共聚焦法测定单个心室肌细胞[Ca2+]i的荧光强度，观测GST对分离豚鼠心室肌细胞内游离钙离子浓度（[Ca2+]i）的影响。（5）应用全细胞膜片钳技术，观察GST对豚鼠心室肌细胞L-型钙通道电流（ICa-L）的影响。1 三羟异黄酮对离体家兔股动脉张力的影响及其机制植物雌激素三羟异黄酮 (genistein, GST)使离体的预先收缩的动脉<WP=5>舒张，其舒张的机制仍然不完全清楚。本研究目的是观察植物雌激素三羟异黄酮对离体家兔股动脉的作用及其机制。实验结果如下：（1）在苯肾上脉素（PE ，1 μmol/L）引起血管收缩的基础上，GST（10~40 μmol/L L）剂量依赖性地舒张家兔股动脉；（2）去除血管内皮显著地抑制GST 引起的舒张；（3）在内皮完整情况下，预先应用NOS抑制剂L-NAME（100 μmol/L）也可显著地抑制GST引起的舒张，提示GST的舒血管作用是内皮依赖的,并与一氧化氮有关；（4）在内皮完整的和去除内皮的股动脉环，预先应用L-型钙通道激动剂，Bay K 8644（0.5 μmol/L）也显著抑制由GST 引起的血管舒张。以上结果表明，GST引起的兔股动脉的舒张是部分内皮依赖的，且与拮抗钙有关。2 区域性血管床对局部注射植物雌激素三羟异黄酮的反应在72只麻醉大鼠，分别采用后肢、肾脏和肠系膜动脉在体恒流灌注法，观察了向灌流环路中直接注射植物雌激素三羟异黄酮（genistein, GST）的血管效应，以灌流压的增减反映血管的收缩和舒张。实验结果如下：（1）不同剂量的GST（0.4、0.8、1.2 mg/kg）注射于股部灌注环路时，剂量依赖性地降低股动脉的灌流压。GST的这一效应可被L-硝基精氨酸甲酯（L-NAME）部分阻断，预先注射蛋白酪氨酸磷酸酶抑制剂正钒酸钠 (50 μg/kg)，则可部分抑制GST（0.8 mg/kg）引起的效应；（2）向肾血管灌注环路中直接注射 GST， 也可剂量依赖性地降低肾动脉的灌流压。预先注射正钒酸钠可完全抑制GST引起的效应。而L-NAME对此效应无影响；（3）向肠系膜血管灌流环路中注射GST，可剂量依赖性地降低其灌流压，此效应可被正钒酸钠部分抑制，而L-NAME对其无影响。根据上述结果得出的结论是：GST降低后肢，肾脏和肠系膜血管床的血管张力，其机制与酪氨酸激酶抑制有关，而在股动脉还与NO释放有部分关系。3 植物雌激素三羟异黄酮对心肌缺血/再灌注损伤及凋亡的影响 本实验的目的在于研究植物雌激素genistein (GST) 对家兔心肌缺血/再灌注损伤及凋亡的影响。阻断家兔心脏左冠状动脉前降支45分钟，再灌注180分钟以引起心肌缺血/再灌注损伤，在心肌缺血前5分钟静脉注射GST (1.0 mg/kg)，观测血流动力学数据,梗死范围和心肌细胞凋亡。实验结果如下：（1）在心肌缺血/再灌注期间,心率，平均动脉压，心肌耗氧量和左心室±dp/dtmax显著降低；（2）损伤对照组心肌梗死范围占缺血<WP=6>心肌的60.23±3.97%，而GST组心肌梗死范围只占缺血心肌的39.62±4.3% (P<0.01)；（3）琼脂糖凝胶电泳显示损伤对照组心肌DNA呈云梯状改变，而在GST组则无此改变；（4）与损伤对照组比较，GST组缺血心肌凋亡细胞明显减少(TUNEL染色)；（5）流式细胞仪测定损伤对照组及GST组缺血心肌凋亡率分别为15.33±1.33%和3.88±0.33%。损伤对照组的缺血心肌Fas和Bax蛋白表达比GST组的高(P<0.01)，而Bcl-2/Bax比率在GST组较损伤对照组的为高(P<0.01)。根据以上结果得出的结论是：植物雌激素GST可减少家兔心肌梗死范围和细胞凋亡。4 三羟异黄酮对豚鼠心室肌细胞内游离钙浓度的影响用激光共更多还原

【Abstract】 Phytoestrogens are naturally occurring, derived from soybeans dephenolic compounds that are similar in structure and function to estrodiol, and bind to estrogen receptors and have estrogen-like cardiovascular effects. Major categories of phytoestrogens include isoflavones, lignans and coumestans. Genistein (GST) is classified as one of the isoflavones. A large body of evidence indicates that phytoestrogens, including GST, may confer cardiovascular protection. For instance, GST could play a role in preventing arteriosclerosis, inhibiting proliferation of vascular smooth cells and angiogenesis, relaxing arteries, thus, it had protective action on cardiovascular system. Our previous studies demonstrated that GST reduced duration of action potential (APD) in normal papillary muscles. In partially depolarized papillary muscles, GST not only reduced APD, but also decreased the amplitude of action potential (APA), overshoot (OS) and maximal velocity of phase 0 depolarization (Vmax); Pretreatment with GST markedly inhibited delayed afterdepolarization (DAD) and triggered activity (TA) induced by ouabain in a concentration-dependent manner. GST reduced maximal rate of depolarization, the velocity of diastolic (phase 4) depolarization (VDD), rate of spontaneous firing (RSF), and the amplitude of action potential in a concentration-dependent manner in sinoatrial node and atrioventricular node pacemaker cells of rabbits. GST also decreased the velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF) in human artrial fibers. However, little is known about other cardiovascular actions of GST. The purposes of the present study were to observe: (1) Action of genistein on tension of isolated femoral artery and its mechanism in rabbits; (2) Responses of regional vascular beds to local injection of genistein in rats; (3) Effects of phytoestrogen genistein on myocardial ischemia/reperfusion injury and apoptosis in rabbits; (4) Effects of genistein on intracellular free calcium <WP=9>concentration in guinea pig ventricular myocytes; (5) Effect of genistein on L-type calcium current in guinea pig ventricular myocytes.1 Action of genistein on tension of isolated femoral artery and its mechanism in rabbits The phytoestrogen genistein has been shown to relax preconstricted arteries in vitro; the mechanism of this relaxation remains incompletely understood. The present study aimed to investigate the effect of phytoestrogen genistein on the tension of rabbit femoral arteries in vitro and to determine the mechanism of such relaxation. The results are as follows: (1) Genistein (10 ~ 40 μmol/L) relaxed femoral arterial rings in a concentration-dependent manner under the condition of precontraction induced by phenylephrine (PE, 1μmol/L); (2) Removal of the endothelium significantly inhibited genistein-induced relaxation; (3) Pretreatment with NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 μmol/L) also significantly inhibited this relaxation by genistein, implying that the concentration-dependent vasorelaxation caused by genistein is endothelium-dependent and involved nitric oxide; (4) Pretreatment with an L-type calcium channel agonist, Bay K 8644 (0.5 μmol/L), also significantly inhibited the genistein-induced relaxation in both endothelium-intact and endothelium-denuded rings. The results suggest that the genistein-induced vascular relaxation of the rabbit arteries is partially endothelium-dependent and involves calcium antagonistic mechanism.2 Responses of regional vascular beds to local injection of genistein in rats The effects of local injection of genistein on femoral, renal, and mesenteric vascular beds were investigated respectively by constant flow perfusion method in 72 anaesthetized rats. The results are as follows: (1) Genistein (0.4, 0.8, 1.2 mg/kg) decreased the perfusion pressure (PP) of femoral vascular bed in a dose-dependent manner. The effect of genistein (0.8 mg/kg) was partially inhibited by L-NAME, or by sodium orthovanadate (50μg/kg), a potent inhibitor of protein更多还原