【作者】 韩黎黎；

【导师】 陈志武；

【作者基本信息】 安徽医科大学 ， 药理学， 2011， 硕士

【摘要】 内皮衍生超极化因子（endothelium dependent hyperpolarizing factor , EDHF）是由内皮细胞产生的非一氧化氮（nitric oxide, NO）、非前列环素（prostacyclin , PGI2 ）的一类具有舒血管作用的物质。研究表明在多种属﹑多部位的血管如人、犬冠状动脉﹑小鼠肠系膜动脉以及猪冠状动脉中均发现存在EDHF。它们和内皮素、血栓素A₂等血管收缩因子协同作用,调节局部血管紧张度和血流量。在缺氧、酸中毒等病理情况下,EDHF通过舒张血管对器官血液供应的维持有着重要的意义。但在脑缺血模型脑血管中,EDHF化学本质尚不清楚。映山红花总黄酮（total flavones of rhododendra, TFR）是从杜鹃科植物映山红花中提取的有效成分。前期研究表明TFR具有止咳、祛痰,镇痛,抗炎,心肌缺血保护作用。张建华在实验研究中表明TFR对正常大鼠脑基底动脉具有舒张作用。本实验探讨在大鼠全脑缺血模中,离体大鼠脑基底动脉血管舒张作用有无EDHF反应,并研究TFR的EDHF作用及其可能的机制。目的:1.观察TFR诱导的全脑缺血再灌大鼠脑基底动脉产生EDHF介导的平滑肌细胞的超级化作用和舒张作用;2.探讨TFR抗大鼠脑缺血再灌注损伤作用的EDHF机制及与钾离子通道的关系。方法:1.四血管结扎法建立大鼠全脑缺血模型。2.采用细胞内电位记录实验方法,观察并测定血管平滑肌细胞膜电位超极化的变化。3.采用离体血管舒缩功能测定法,将大鼠脑基底动脉置于灌流浴槽中,观察并测量血管直径的变化。4.急性消化分离基底动脉平滑肌细胞,全细胞膜片钳法记录大鼠基底动脉平滑肌细胞钾电流的变化。结果:1四血管结扎法造大鼠全脑缺血模型,脑缺血时大鼠的脑电图幅度迅速下降,再灌注后脑电图的幅度逐渐恢复。分析大鼠脑电图,四血管结扎法成功建造大鼠全脑缺血模型。2在实验中,应用NO合酶抑制剂(L-NAME,3×10^-5mol/L)和PGI₂合酶抑制剂(Indomethacin,10^-5mol/L), ACh可引起全脑缺血再灌大鼠CBA平滑肌细胞产生浓度依赖性的显著的平滑肌细胞超极化作用和血管舒张作用;3在实验中,应用NO合酶抑制剂（L-NAME,3×10-5mol/L）和PGI2合酶抑制剂(Indomethacin,10^-5mol/L),不同浓度的TFR均可诱导全脑缺血再灌大鼠CBA平滑肌细胞产生浓度依赖性的平滑肌细胞超极化作用和血管舒张作用。4全细胞膜片钳记录平滑肌细胞钙激活的钾电流实验中发现,TFR（300 2700 mg/L）能使胶原酶消化的全脑缺血大鼠基底动脉平滑肌细胞的KCa外向电流呈现浓度依赖性的增强。结论:1.全脑缺血再灌模型可明显增强大鼠CBA中非NO、非PGI₂介导的平滑肌细胞超级化和血管舒张,即EDHF反应,的敏感性。2.映山红花总黄酮TFR能明显地诱导全脑缺血再灌大鼠CBA产生浓度依赖性的非NO、非PGI₂介导的血管平滑肌细胞超级化作用和血管舒张作用,即EDHF反应;3. TFR介导的全脑缺血再灌大鼠CBA的EDHF反应可以被内源性H₂S合成酶CSE的阻断剂PPG所抑制,提示其TFR介导的EDHF反应可能涉及到硫化氢的生成;4. TFR介导的全脑缺血再灌大鼠CBA的EDHF反应机制涉及K通道的作用。5.映山红花总黄酮TFR作为抗脑缺血再灌注损伤的新药开发有潜在的临床应用价值。更多还原

【Abstract】 The vascular endothelium releases endothelium-derived relaxing factors in response to some agonists or physical stimuli that mediate relaxation. These factors include nitric oxide （NO）, prostacyclin （PGI2）, and several factors called endothelium-derived hyperpolarizing factors （EDHFs）. At present, the role of EDHF has been researched in various peripheral vascular beds including coronary artery of human and dog , mesenteric artery of rats.Total flavones of rhododendra （TFR） was extracted from the flower of total flavones of Rhododendra. The essential components of TFR are hyperin, quercetin and other flavonoids. It has been preliminary assessed to be of protection against myocardial and cerebral ischemia. In this study, it was studied that EDHF-mediated responses of relaxation and hyperpolarization of vascular smooth muscle cell （VSMC） induced by TFR in rat cerebral basilar artery （CBA） subjected to cerebral ischemia/reperfusion （I/R） .Purpose:1. To study the EDHF-mediated hyperpolarization of VSMC and vasorelaxtion relaxation induced by TFR in rat CBA subjected I/R.2. To investigate the effects of TFR on outwardand K+ currents in rat CBA VSMC.Method:1. Cerebral ischemia reperfusion was induced by 4-vessle occlusion （4-VO） in rat.2. Isolated rat CBA segmnts were suspended in baths containing PSS solution, the relaxation was observed by using measurement of vessel diameter.3. Transmembrane potentials were recorded to evaluate the hyperpolarization effects in rat CBA VSMC. 4. Whole-cell patch clamp recording was used to measure the Ca2+-activated potassium （Kca） current in rat CBA VSMC.Results:1. In the presence of 3×10^-5 mol/L Nω-nitro-L–arginine-methyl-ester （L-NAME, an inhibitor of nitric oxide synthase） and 1×10^-5 mol/L indomethacin （Indo, an inhibitor of PGI₂ synthesis）, the globa cerebral I/R markedly enhanced 1×10-7 1×10^-5 mol/L acetylcholine-elicited hyperpolarization of RMP of VSMC and vasorelaxation in rat CBA,respetively.2. In the presence of L–NAME and Indo, 11 2700 mg/L TFR induced significant and dose-dependent hyperpolarization of RMP of VSMC and vasorelaxation in rat CBA subjected to global cerebral I/R.3. The hyperpolarization and relaxation were obviously inhibited by tetraethylammonium （an inhibitor of IKCa at 1 mmol/L） and 1×10-4 mol/L DL-propargylglycine, an inhibitor of endogenous hydrogen sulfide （H₂S） synthase.4. 300²700mg/L TFR markedly enhanced the Ca2+-activated potassium （Kca） current in rat CBA VSMC.Conclusions:1. Global cerebral I/R could enhance the non-NO-non-PGI₂–mediated responses of hyperpolarization and vasorelaxation in rat CAB.2. In rat CAB subjected to global cerebral I/R, TFR significantly induced EDHF-mediated the responses of hyperpolarization and relaxation, and the response may be related to endogenous H₂S.更多还原