【作者】 许波华；

【导师】 许立；

【作者基本信息】 南京中医药大学 ， 药理学， 2012， 硕士

【摘要】 目的：观察丹酚酸B (Salvianolic acid B,简称Sal B)体外对人脐静脉内皮细胞(HUVECS)增值、迁移和管腔形成的影响,SalB对心肌缺血大鼠血管新生的影响及作用机制；观察SalB的血管舒张作用并探索其作用机制。方法：1.(1)取对数期生长的人脐静脉血管内皮细胞(HUVECS),用MTT法观察SalB对HUVECS活力和增殖的影响；用3H掺入法检测SalB对HUVECS DNA合成率的影响；细胞划痕法检测Sal B对细胞迁移距离的影响；Transwell实验验证Sal B对细胞划痕迁移的作用；用体外毛细血管管腔结构实验检测Sal B对HUVECS管腔形成的影响；(2)将HUVECS用Sal B处理12h、24h、48h后,酶联免疫吸附测定法(ELISA)测定HUVECS上清中VEGF的含量；用试剂盒测定HUVECS上清中NO和NOS的含量；将HUVECS用SalB处理24h和48h后收集细胞,Western Blot检测HUVECS中VEGF的表达；(3)结扎大鼠冠状动脉左降支建立大鼠心肌缺血模型,每组大鼠分别尾静脉注射SalB1.6、3.2和6.4mg/kg,空白组,假手术组和模型组给予同等体积的生理盐水。测定给药后血清VEGF的含量；测定心肌组织匀浆中NO、 NOS、 VEGF的含量；测定缺血心肌的梗死面积；取心脏缺血区域做组织病理学检测；通过免疫组化法测定梗死边缘区新生血管数(MVC)。2.制备大鼠离体胸主动脉环,通过给药观察SalB对离体胸主动脉环的直接作用；(1)观察Sal B对NE收缩血管环的影响：①先加入10-6mol/L NE收缩血管,再加入不同浓度的SalB观察其对收缩血管的舒张能力；②用不同浓度的SalB对血管预处理15min再加入10-6mol/LNE收缩血管,观察最大收缩张力；③用累积加药法加入不同浓度的NE,记录不同浓度NE的收缩曲线,然后冲洗血管,待稳定后,先加入不同浓度的Sal B预处理,然后累积加入不同浓度的NE,记录其收缩曲线,观察其收缩曲线的变化；(2)观察SalB对KCl收缩血管环的影响：①先加入60mmol/L KCl收缩血管,再加入不同浓度的SalB观察其对收缩血管的舒张能力；②先用不同浓度的Sal B对血管预处理再加入60mmol/L KCl收缩血管,观察最大收缩张力；③用累积加药法加入不同浓度的KCl,记录不同浓度KCl的收缩曲线,然后冲洗血管,待稳定后,先加入不同浓度的Sal B预处理,然后累积加入不同浓度的KCl,记录其收缩曲线,观察其收缩曲线的变化；(3)观察Sal B对CaCl2收缩血管环的影响：先通过累积加入不同浓度CaCl2得到CaCl2收缩曲线,然后用不同浓度SalB预处理后再加入不同浓度CaCl2得到CaCl2收缩曲线,观察SalB对收缩曲线的变化；(4)观察Sal B对NE引起的依赖于细胞外钙和内钙的收缩反应的影响；(5)通过加入不同的抑制剂来探讨Sal B舒张血管环作用是否与内皮和钾离子通道有关；(6)通过加入β受体阻断剂来观察SalB舒血管作用是否与p受体有关。结果：1：(1)3H掺入法,结果显示SalB在一定浓度范围内对HUVECS增殖作用明显；细胞划痕实验和Transwell结果表明Sal B对HUVECS迁移有明显的促进作用；管腔形成实验显示,SalB作用HUVECS后可以使管腔形成数增多,管腔长度增长；(2)HUVECS经SalB处理后,HUVECS上清中VEGF、 NO和NOS含量增加,VEGF表达上调；(3)Sal B可以使心肌组织中NO、NOS和VEGF含量增加,同时血清中VEGF含量也增加；Sal B能够减少心肌梗死面积,增加缺血区域和非缺血.区域的血管新生。2：直接加入SalB后发现SalB对基础状态下胸主动脉无明显的直接作用；SalB能明显抑制NE的收缩,使NE的收缩曲线右移；SalB对KCl的收缩无明显影响；SalB能使无钙高钾中CaCl2引起的收缩曲线明显右移；经无钙液处理后SalB能阻断NE诱发的外钙收缩和内钙收缩；SalB舒张血管作用与内皮、钾离子通道和β受体无关。结论：(1)SalB在体外能促进HUVECS的增值、迁移和管腔形成,其作用机制可能与上调HUVECS中VEGF的表达有关；(2)SalB能促进心肌缺血大鼠心脏的血管新生,对缺血心肌具有保护作用；(3)SalB可呈非内皮依赖性舒张血管,其作用机制可能与阻断受体依赖性钙通道、电压依赖性钙通道引起的外钙内流和阻断三磷酸肌醇(IP3)受体引起的内钙释放有关,与血管平滑肌上钾离子通道和β受体无关。更多还原

【Abstract】 Aim:To observe the effect of salvianolic acid B (Sal B) on proliferation, migration and tube formation in vitro, to explore the protective effects of Sal B of myocardial angiogenesis and molecular mechanism rats with acute myocardial ischemia; to investigate the vasodilatory effect of Sal B and its mechanisms.Methods:1(1) MTT method was used to determine the effect of Sal B on the proliferation of HUVECS. Tritium-labeled thymidine method was used to observe the effect of Sal B on the DNA synthesis about HUVECS. HUVECS migrations were detected by scratch method and Transwell assay. Capillary was detected by Matrigel-plug assay in vitro.(2) Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of VEGF in serum of HUVECS after treated with Sal B for12h、24h and48h, and then use kits to detect the content of NO and NOS. Western Blot was used to determine the effect of Sal B on the expression of VEGF in HUVECS which were treated with Sal B for24h and48h.(3) Myocardial ischemia was made by ligation of left anterior descending branch of coronary artery. Experimental animals were randomized to7groups, control group, sham group, model group, VEGF group and Sal B(1.6、3.2、6.4mg/kg) groups, the drugs were injected through the tail vein for14days. After treatment, VEGF blood serum and the homogenate and nitric oxide (NO) and nitric oxide synthase (NOS) in homogenate of myocardial tissue were measured. Meanwhile, the infarct area of ischemia myocardial and the pathological changes of myocardial tissue were also observed. Microvessel counting (MVC) was detected by immunohistochemical method.Making the thoracic aortae rings of rats in vitro. To observe the vasodilator effect of Sal B on thoracic aortae rings of rats in vitro;(1) To determine the effects of Sal B on the contractions induced by noradrenaline (NA) in thoracic aortae rings.①Adding NE(10-6mol/L) to contract the thoracic aortae rings, and then add Sal B with different concentrations to observe the level of relaxation.②Adding Sal B with different concentrations to incubate with the thoracic aortae rings for15min, and then add NE(10-6mol/L) to obverse the maximum contraction.③The dose-effect curve of the thoracic aortae ring’s contraction was obtained by adding an accumulative series concentrations of NE. Then Sal B was added after the strain of the thoracic aortae rings recuperated, measured the NE’s dose-effect curve to observe the relaxation level of the thoracic aortae rings;(2) To determine the effects of Sal B on the contractions induced by potassium chloride (KCl) in thoracic aortae rings.①Adding KCl (60mmol/L) to contract the thoracic aortae rings, and then add Sal B with different concentrations to observe the level of relaxation.②Adding Sal B with different concentrations to incubate with the thoracic aortae rings for15min, and then add KCl (60mmol/L) to obverse the maximum contraction.③The dose-effect curve of the thoracic aortae ring’s contraction was obtained by adding an accumulative series concentration of KCl. Then Sal B was added after the strain of the thoracic aortae rings recuperated, measured the KCl’s dose-effect curve to observe the relaxation level of the thoracic aortae rings;(3) The dose-effect curve of the thoracic aortae ring’s contraction was obtained by adding an accumulative series concentrations of CaC2. Then Sal B was added after the strain of the thoracic aortae rings recuperated, measured the CaCl2’s dose-effect curve to observe the relaxation of the thoracic aortae rings;(4) To observe the effect of Sal B on the contraction of the thoracic aortae rings depending on the intracellular calcium and extracellular calcium;(5) By adding different inhibitors to investigate whether the vasorelaxant effects of Sal B were related to the endothelium and K+channels;(6) By adding P-blocker to observe whether the vasorelaxant effect of Sal B was related to the (3receptor.Results:1(1)3H-TdR assay showed that Sal B promoted the proliferation of HUVECS. The scratch method and Transwell assay showed that Sal B accelerated the migration of HUVECS. The tube formation assay showed that the tube number and the length of the tube were higher than control group.(2) Sal B can improve the content of NC、NOS and VEGF in supernatant released by HUVECS, the expression of VEGF also can be up-rcgulated.(3) Sal B could decrease the infarct area in ischemia myocardium and increase the content of NO、NOS and VEGF in the myocardial tissue, the content of VEGF in plasma can also be increased. Sal B could promote angiogenesis in ischemic regional and non-ischemia regional.2(1) There was no direct affect to the thoracic aortae rings for Sal B.(2) Sal B can inhibit the contraction of NE significantly and cause the rightward shifting of the dose-response curve of NE. Sal B had no significant effect on the contraction of KCl. In Ca2+free and high K+solution Sal B can cause the rightward shifting of the dose-response curve of CaCl2. Sal B can inhibit the contraction induced by extracellular calcium and intracellular calcium which were caused by NE. The vasorelaxant effects of Sal B were not related to the endothelium、 K+channels and the β receptor. Conclusions:(1) Sal B promotes the proliferation, migration and tube-like structure formation. Its mechanism may be related to the up-regulation of VEGF expression.(2) Sal B can enhances angiogenesis after myocardial infraction and protect the ischemia myocardium in ischemic myocardium of rats.(3) Sal B can relax rat thoracic aorta rings without endothelium. The mechanisms may involve the reduction in Ca2+influx through the receptor-dependent pathway、 voltage-dependent pathway and block the IP3receptor-induced Ca2+release, but not correlated with the K+channels and the β receptor.更多还原