白果内酯经血小板活化因子受体实现对大鼠心脏的抗缺血效应

论文摘要

许多研究已经证实,从银杏中提取的萜烯三内酯对脑和心脏有抗氧化和抗缺血效应,但这些效应的具体机理尚不清楚。本文研究了一种从银杏中提取的萜类成分——白果内酯,在缺氧情况下对大鼠缺血心脏的保护作用,同时证实,血小板激活因子受体（PAFR）和血小板激活因子乙酰水解酶（PAF-AH）参与白果内酯对大鼠缺血心脏的保护作用。血小板激活因子（PAF）的通路在缺氧情况下十分重要,调节PAF信号转导通路可能避免或减轻心肌梗死的程度。实验中,我们以新生SD大鼠的心肌细胞和内皮细胞为研究对象,预先给予不同浓度（0.5-500 ng/ml）的白果内酯,将上述细胞置于缺氧环境中后,观察细胞的存活率,并提取蛋白质与核糖核酸（RNA）,分别进行蛋白质印迹（Western Blotting）与逆转录聚合酶链式反应（RT-PCR）检测PAF通路的情况大鼠缺血心脏的保护作用。此外,我们采用大鼠心肌梗死的在体模型证实了白果内酯的心肌保护作用,及PAFR与PAF-AH的参与。在两批在体实验中,第一次在心肌梗死前应用白果内酯腹腔注射,着重考察白果内酯的心脏保护作用；第二次着重考察药物对心肌梗死后心力衰竭的治疗作用。为了评价两次实验中白果内酯的心肌保护作用,主要检测了死亡率梗死面积、肺脏湿重/体重比、体重、超声心电图,以及利用基于同步辐射的冠状动脉造影术得到的冠状动脉图像。实验结果显示,白果内酯对心肌细胞和内皮细胞的保护作用呈浓度依赖性,其中5ng/ml的白果内酯显著降低了心肌细胞和内皮细胞的死亡率。PAFR的mRNA在缺血细胞表达上调,但经过白果内酯处理过的心肌细胞,下调PAFR mRNA的表达,与正常对照组比较,差异无统计学意义。在缺血组织上,PAFR蛋白质表达上调,但白果内酯（10 mg/kg i.p.）处理过的组具有减少其蛋白表达的作用。两次实验并没有给出令人信服的证据说明,白果内酯在动物模型上也有心肌保护效果,该部分实验还有待于进一步证实。本文通过实验证明：心肌细胞在缺氧过程中,血小板激活因子（PAF）及其受体参与其中,且白果内酯可能与该受体作用,在离体条件下产生了心肌保护效果。后续实验应着重研究白果内酯的在体的心肌保护效果存在的正确条件。

论文目录

• 摘要
• Abstract
• CONTENTS
• Table of Abbreviations
• List of Figures
• List of Tables
• 1 INTRODUCTION
• 1.1 Ginkgo biloba
• 1.2 Extract Ginkgo biloba and its constituents
• 1.3 The terpene trilactones ginkgolide A, B and bilobalide
• 1.4 Myocardial infarction
• 1.5 PAF and PAFR
• 1.6 EGb761 and endothelial cells/angiogenesis
• 1.7 Angiogenesis
• 1.8 Coronary angiography by synchrotron radiation
• 1.9 Aim of the study
• 2 MATERIALS AND METHODS
• 2.1 Materials
• 2.1.1 Ginkgolide A, B and bilobalide
• 2.1.2 Aspirin
• 2.1.3 Erythropoietin
• 2.1.4 Solutions and Laboratory equipment
• 2.1.5 Cell culture solutions
• 2.1.6 Animals
• 2.2 Methods
• 2.2.1 NC culture
• 2.2.2 HUVEC culture
• 2.2.3 Hypoxia
• 2.2.4 MTT assay
• 2.2.5 Lactate dehydrogenase activity （LDH） assay
• 2.2.6 Hoechst staining
• 2.2.7 Immunofluorescence
• 2.2.8 Flow cytometry
• 2.2.9 Myocardial Infarction model
• 2.2.10 Trial 1:Acute myocardial infarction
• 2.2.11 Trial 2:heart failure
• 2.2.12 Infarct size
• 2.2.13 Lung-to-body weight ratio
• 2.2.14 Coronary angiography
• 2.2.15 Doppler Echocardiography
• 2.2.16 Protein quantification by Bicinchoninacid （BCA）
• 2.2.17 Phospholipase A2 activity assay
• 2.2.18 RNA isolation and RT-PCR
• 2.2.19 Western blotting
• 2.2.19.1 In Vitro Sample Preparation
• 2.2.19.2 In Vivo Sample Preparation
• 2.2.19.3 Protein separation
• 2.2.19.4 Transfer
• 2.2.19.5 Protein staining
• 2.2.20 Statistical analysis
• 3 RESULTS
• 3.1 In vitro:Cell culture experiments
• 3.1.1 MTT
• 3.1.1.1 NC
• 3.1.1.2 HUVEC
• 3.1.2 LDH
• 3.1.3 PLA2 activity
• 3.1.4 Hoechst staining
• 3.1.4.1 NC
• 3.1.4.2 HUVEC
• 3.1.5 Apoptosis detection by FACS HUVEC
• 3.1.6 Apoptosis markers bax, bcl-2 and apaf-1 mRNA expression in NC and endothelial cells （RT-PCR）
• 3.1.6.1 NC
• 3.1.6.2 HUVEC
• 3.1.7 Western blotting of apoptosis marker proteins bax and bcl-2
• 3.1.8 Immunofluorescence microscopy of PAFR on NC
• 3.1.9 PAFR and PAFAH mRNA expression （RT-PCR） in NC
• 3.1.10 Western blotting of PAFR and PAFAH in NC
• 3.2 In vivo:Myocardial Infarction model
• 3.2.1 Trial 1:Protective effects of bilobalide during myocardial infarction
• 3.2.2 Trial 1:Western blotting of apoptosis marker proteins bax and bcl-2 in myocardial tissue
• 3.2.3 Trial 1:Western blotting of PAFR and PAFAH in myocardial tissue
• 3.2.4 Trial 2:Curative effects of bilobalide in heart failure following myocardial infarction
• 3.2.5 Trial 2:Coronary Angiography using synchrotron radiation
• 3.2.6 Trial 2:Doppler Echocardiography Studies
• 4 DISCUSSION
• 4.1 In vitro
• 4.1.1 Bilobalide and ginkgolide A improve viability of NC during hypoxia
• 4.1.2 Bilobalide improves viability of endothelial cells during hypoxia
• 4.1.3 Bilobalide reduces LDH leakage during hypoxia
• 4.1.4 Bilobalide reduces PLA2 activity during hypoxia
• 4.1.5 Bilobalide doesn't affect apoptosis marker proteins in NC during hypoxia
• 4.1.6 Bilobalide inhibits apoptosis in endothelial cells during hypoxia
• 4.1.7 NC express PAFR, while fibroblasts do not
• 4.1.8 Bilobalide downregulates PAFR expression in NC during hypoxia
• 4.1.9 Bilobalide upregulates PAF-AH expression in NC during hypoxia
• 4.2 In vivo
• 4.2.1 Bilobalide doesn't affect mortality after myocardial infarction
• 4.2.2 Ginkgolide A enhances myocardial infarction
• 4.2.3 PAFR is upregulated in the infarcted left ventricle and bilobalide downregulates PAFR expression in the infarcted left ventricle
• 4.2.4 PAF-AH is downregulated in the in the infarcted left ventricle and bilobalide upregulates PAF-AH after myocardial infarction
• 4.2.5 Bilobalide doesn't affect mortality in heart failure after myocardial infarction
• 4.2.6 Bilobalide doesn't affect functional parameters of the left ventricle in heart failure
• 4.2.7 Coronary angiography using synchrotron radiation is an interesting tool to observe angiogenesis in vivo
• 5 CONCLUSION
• REFERENCES
• APPENDIX
• Curriculum vitae
• Acknowledgments