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1、 P2X3受体论文：黄芩苷对颈部交感神经节细胞P2X_（2/3）受体介导心肌缺血引发交感兴奋反射的作用研究【中文摘要】和冠心病、心肌梗死是临床常见的疾病,心肌缺血时心肌细胞等释放大量ATP至细胞外或细胞间隙刺激心脏感觉传入神经,加强交感兴奋反射活动,导致颈部交感神经节节后交感传出神经兴奋性增强,从而引起心率加快、血压升高,加重心肌损伤。ATP激活P2X受体产生效应。我们实验室前期研究结果显示,颈部交感神经节的P2X3和P2X2/3受体在心肌缺血伤害性感觉传递中起重要作用。黄芩苷,是从唇形科植物黄芩的干燥根中提取的一种黄酮类化合物,具有抗炎、抗氧化、抗病毒和抗癌的性质。近些年,广泛的研究证实了黄
2、芩苷对许多疾病具有治疗效果,是一种重要的临床药物。但是,黄芩苷对心肌损伤的保护机理尚未明确,是否可作用于颈部交感神经节神经细胞P2X2/3受体影响心肌缺血引发的交感增强反射对心肌产生保护作用目前尚未见文献报道。本实验拟应用心肌缺血大鼠模型,观察黄芩苷处理后大鼠血压、心率的变化,以及对颈上、星状交感神经节细胞P2X2/3受体表达的影响,了解黄芩苷在心肌缺血引发的交感反射中的作用及其可能机制。方法：（1）结扎大鼠左冠状动脉前降支建立心肌缺血动物模型,通过心电图、心肌HE染色观察黄芩苷对心肌缺血损伤大鼠心功能及形态的影响；（2）使用无创法血压计测量大鼠的血压、心率,观察黄芩苷处理的心肌缺血大鼠血压、
3、心率的变化,并与对照组比较；（3）检测血清肌酸激酶（CK-MB）和心肌肌钙蛋白T（cTn-T）两种心肌酶含量,观察黄芩苷对心肌缺血损伤的作用；（4）免疫荧光双标法观察心肌缺血模型组、心肌缺血模型黄芩苷处理组及对照组颈上、星状交感神经节细胞及心肌的P2X3、P2X2受体的共表达情况。（5）Western Blot和免疫组织化学法观察心肌缺血模型组、心肌缺血模型黄芩苷处理组及对照组的P2X3、P2X2受体的表达情况。（6）RT-PCR观察心肌缺血模型组、心肌缺血模型黄芩苷处理组及对照组颈上、星状交感神经节细胞及心肌的P2X3、P2X2受体mRNA表达的变化。（7）ELISA的方法检测各组大鼠血清中
4、肾上腺素浓度的变化；（8）CellTiter-Glo（?）发光法检测各组大鼠ATP浓度的变化。结果：（1）结扎大鼠左冠状动脉前降支,心电图立即出现ST段呈弓背向上抬高,模型组14天后心电图出现深大的病理性Q波,HE染色显示明显出血,心肌萎缩、纤维化甚至出现坏死灶,外观上可见代偿性肥大和大面积的结缔组织增生现象。然而,黄芩苷处理后大鼠心电图的异常变化和心肌损伤明显改善。（2）心肌缺血大鼠收缩压、舒张压、心率与正常对照组相比均增加（n=5）（p0.05）。黄芩苷处理组大鼠收缩压、心率略高于正常组、假手术组和黄芩苷对照组（n=5）（p0.05）。（4）免疫荧光双标染色结果显示,大鼠颈上、星状交感神经
5、节细胞和心肌组织存在P2X3受体和P2X2受体的共表达,心肌缺血模型组表达明显高于对照组,黄芩苷处理心肌缺血后,P2X3受体和P2X2受体表达明显下降。（5）免疫组织化学染色结果显示,心肌缺血组大鼠颈上、星状交感神经节细胞和心肌组织P2X3受体和P2X2受体表达高于正常组、假手术组、黄芩苷对照组和黄芩苷处理组（p0.05）。（6）RT-PCR结果显示,心肌缺血模型组大鼠颈上、星状交感神经节细胞和心肌组织P2X3受体和P2X2受体mRNA的表达高于正常组、假手术组、黄芩苷对照组和黄芩苷处理组,黄芩苷处理心肌缺血后,灰度值明显降低（p0.05）。（9）CellTiter-Glo（?）发光法检测各组
6、大鼠血清中ATP浓度结果显示,心肌缺血组血清中ATP含量高于正常组、假手术组、黄芩苷对照组和黄芩苷处理组（p0.05）。结论：黄芩苷可以降低心肌缺血大鼠颈部神经节神经细胞及心肌交感神经P2X3和P2X2受体表达,从而减轻心肌缺血引发的血压升高、心率加快。颈上星状神经节细胞P2X2/3受体介导心肌缺血引发的交感兴奋反射,黄芩苷减轻心肌缺血组大鼠颈上、星状交感神经节细胞和心肌组织P2X3受体和P2X2受体mRNA和蛋白表达,表明黄芩苷可能通过作用于P2X2/3受体影响心肌缺血损伤引发的交感兴奋反射对心脏产生保护作用。【英文摘要】Backround and :Coronary heart disea
7、se and myocardial infarction is clinically common diseases. During myocardial ischemia, a number of cell types release large quantities of ATP in the extracellular or interstitial space, which activates cardiac afferent nerve, characterized by an increase in blood pressure and sympathetic nerve acti
8、vity. The strengthen sympathoexcitatory reflex may increase the blood pressure and heart rates, which enhanced ischemic myocardiaol injury. ATP activates P2X receptors to produce functions. Previous research works in our laboratory showed that P2X3 and P2X2/3 receptors of cervical sympathetic gangli
9、a contributed to the myocardial ischemic nociceptive response. Baicalin is the major flavonoid components of the root of Scutellaria baicalensis and known for its strong anti-inflammatory properties, anti-oxidant properties, anti-viral properties, anti-cancer properties. It was extensively researche
10、d for utility in a number of therapeutic areas as an important medical agent. It is not clear for the role of baicalin in the sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat cervical sympathetic ganglia. The present study was aimed to observe
11、 the changes of rat systolic blood pressure, heart rate in the myocardial ischemic rats and the changes of P2X2/3 receptor expression value in superior cervical ganglion (SCG), stellate ganglion (SG) neurons and heart after treated with baicalin and to explore the effect mechanisms of baicalin on sy
12、mpathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat SCG and SG neurons.Methods:(1)A rat model of myocardial ischemia injury was established by ligating the left anterior descending coronary artery. Electrocardiogram (ECG) and H-E stain of cardiac t
13、issue were used to observe the effect of baicalin on heart function and cardiac tissue in rat myocardial ischemia injury. (2)The blood pressure and heart rates were measured by non-invasive blood pressure determinator (the indirect tail-cuff plethysmography) to observe the effect of baicalin on the
14、blood pressure and heart rates in rat myocardial ischemia injury. (3)CK-MB and cTn-T(cardiac muscle enzymes) were measured cardiac muscle enzymed to observe the effect of baicalin on myocardial ischemia injury. (4)The coexpressions of P2X3 and P2X2 receptors in rat SCG, SG neurons and heart of diffe
15、rent groups were detected by immunofluorescence. (5)The expression of P2X3 and P2X2 receptors protein in rat SCG, SG neurons and heart were analyzed by western blotting and immunohistochemistry to observe the effect of baicalin on myocardial ischemia injury. (6)The expression of P2X3 and P2X2 recept
16、ors mRNA in rat SCG, SG neurons and heart were analyzed by RT-PCR to observe the effect of baicalin on myocardial ischemia injury. (7)Epinephrine was measured by ELISA. (8)ATP was measured by CellTiter-Glo.Results:(1)After ligating the left anterior descending coronary artery, ST segment in the ECG
17、was high upward. After 14 days, the abnormal Q wave appeared obviously in myocardial ischemic rats. HE stain showed significant bleeding, myocardial atrophy, fibrosis and necrosis. Myocardial tissues in myocardial ischemic group showed severe atrophy, hypertrophy and proliferation of fiber connectiv
18、e conditions. After treated with baicalin, abnormal Q wave and myocardial issue changes induced by myocardial ischemia were improved. (2)Compared with control rats systolic blood pressure, diastolic blood pressure and heart rate in the myocardial ischemic rats were increased (n=5)(p0.05). Systolic b
19、lood pressure, diastolic blood pressure and heart rate in the myocardial ischemic rats treated baicalin were higher than those in control groups(n=5)(p0.05). (4)Double-label immunofluorescence results showed that P2X3 and P2X2 receptors were co-expressed in SCG, SG neurons and myocardial tissues. Th
20、e co-expressed cell of P2X3 and P2X2 receptors in myocardial ischemic injury group exhibited more intense staining than those in control groups. The co-expressed value in the ischemic rats treated baicalin were lower than those in ischemic rats(n=5)(p0.05). In the SCG, SG neurons and myocardial tiss
21、ues, the average optical density (ODs) of P2X2 receptor expression in myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin and control groups(p0.05). (6)The expression value of P2X3 and P2X2 mRNA in SCG, SG neurons and myocardial tissues w
22、as studied by RT-PCR. The average OD of P2X3 mRNA in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury treated with baicalin, normal group, sham group and baicalin control group (p0.05). (9)The concentration of ATP in rat serm is tested by CellTiter-G
23、lo. The concentration of ATP in myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin group and control group (p0.05).Conclusions:Baicalin could decrease the expressions of P2X3 and P2X2 receptor mRNA and protein in cervical sympathetic gan
24、glia and myocardial sympathetic nerves of myocardial ischemia rats. These results suggest that baicalin may decrease sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat cervical sympathetic ganglia to depress blood pressure and heart rate and pro
25、duce the protection of heart.【关键词】P2X3受体 P2X2受体 P2X2/3受体 交感兴奋反射 颈上神经节 星状神经节 黄芩苷【英文关键词】P2X3 receptor P2X2 receptor P2X2/3 receptor sympathoexcitatory reflex superior cervical ganglia stellate ganglia baicalin【目录】黄芩苷对颈部交感神经节细胞P2X_（2/3）受体介导心肌缺血引发交感兴奋反射的作用研究摘要3-6ABSTRACT6-9第1章 引言12-14第2章 材料与方法14-272.1 材
26、料14-192.1.1 动物142.1.2 主要试剂14-152.1.3 主要仪器15-162.1.4 溶液配制16-192.2 方法19-272.2.1 实验动物的分组202.2.2 大鼠心肌缺血模型制备202.2.3 血压、心率测定202.2.4 HE染色20-212.2.5 心肌酶测定212.2.6 免疫荧光双标法212.2.7 免疫组织化学21-222.2.8 蛋白印迹22-232.2.9 RT-PCR23-252.2.10 肾上腺素酶联免疫测定25-262.2.11 ATP测定262.2.12 统计方法26-27第3章 结果27-453.1 血压、心率及心电图的变化27-283.2
27、心肌结构及心肌酶变化28-293.2.1 心肌HE染色283.2.2 心肌酶28-293.3 免疫荧光双标29-313.3.1 颈上神经节荧光双标法(P2X_3和P2X_2受体)29-303.3.2 星状神经节免疫荧光双标30-313.3.3 心肌免疫荧光双标313.4 免疫组织化学31-363.4.1 颈上神经节P2X_3、P2X_2受体免疫组化法31-333.4.2 星状神经节P2X_3、P2X_2受体免疫组化法33-343.4.3 心肌P2X_3、P2X_2免疫组化法34-365.5 RT-PCR36-403.5.1 颈上神经节P2X_3和P2X_2受体的mRNA表达36-373.5.2 星状神经节P2X_3和P2X_2受体的mRNA表达37-383.5.3 心肌P2X_3和P2X_2受体的mRNA表达38-403.6 蛋白印迹40-423.6.1 颈上神经节P2X_3和P2X_2受体的蛋白表达40-413.6.2 星状神经节P2X_3和P2X_2受体的蛋白表达413.6.3 心肌P2X_3和P2X_2受体的蛋白表达41-423.7 ELISA检测大鼠肾上腺素42-443.8 ATP检测44-45第4章 讨论45-47第5章 结论47-48致谢48-49参考文献49-52攻读学位期间的研究成果52-54综述54-63参考文献61-63
