【作者】 杜雪君；

【导师】 雷燕；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2010， 博士

【摘要】 治疗性血管新生已经成为缺血性疾病研究领域的热点,益气活血中药治疗冠心病的历史悠久,疗效显著,其促进血管新生,改善心肌供血,已经通过临床和实验研究得到证实,因此益气活血中药促血管新生可能是其治疗冠心病的重要机制之一。本研究通过探讨益气活血中药促大鼠缺血心肌血管新生的分子机制,揭示其干预血管生成的作用和分子靶标,为其防治缺血性疾病提供实验依据。本论文分为三部分：基础研究、临床文献系统评价和文献综述。一、基础研究包括以下三部分：实验一：益气活血中药对大鼠缺血心肌的促血管生成作用目的：观察益气活血中药人参三七组方促进大鼠缺血心肌血管新生的作用。方法：100只Wistar大鼠随机分为空白组、假手术组、模型组、倍他乐克组和大、小剂量人参三七组方组。采用冠状动脉结扎法建立大鼠急性心梗模型,并于造模后予相应药物治疗12 d。观察各组心肌细胞形态学改变,检测血清血管内皮细胞生长因子(VEGF)的表达；采用Ⅷ因子及CD105抗体检测微血管密度。结果：人参三七组方能够减轻心肌细胞变性,减少炎症细胞,增加微血管密度,提高血清VEGF的浓度。结论：人参三七组方能够保护缺血心肌细胞,具有良好促进急性心梗后大鼠缺血心肌的微血管新生,增加缺血心肌微血管密度的作用。实验二：益气活血中药对大鼠缺血心肌VEGFR-2和HIF-1α表达的影响目的：探讨益气活血中药人参三七组方对AMI大鼠缺血心肌VEGFR-2和HIF-1α表达的影响。方法：造模及处理方法同实验一,给药结束后,蛋白免疫印迹法检测缺血心肌的VEGFR-2和HIF-1α的蛋白表达,实时聚合酶链式反应法检测缺血心肌VEGFR-2、HIF-1αmRNA表达。结果：大、小剂量人参三七组方组及倍他乐克组VEGFR-2、HIF-1α蛋白和mRNA表达均显著高于模型组(P＜0.05)；大、小剂量组之间比较,差异亦有统计学意义。倍他乐克组VEGFR-2、HIF-1α蛋白和mRNA高于大、小剂量人参三七组方组(P<0.01)。结论：人参三七组方能够促进缺血心肌VEGFR-2、HIF-1α表达,从而改善心肌缺血,促进侧支循环的形成,具有一定剂量依赖性。实验三：益气活血中药对Ras下游信号转导通路的影响目的：观察益气活血中药人参三七组方对Ras信号通路的影响,探讨益气活血中药促进血管新生的信号转导途径。方法：造模及处理方法同实验一,给药结束后,蛋白免疫印迹法检测缺血心肌中Ras.c-Raf-1.ERK1／2及p-ERK1／2的蛋白表达,实时聚合酶链式反应法检测缺血心肌Ras. c-Raf-1、ERK1、ERK2 mRNA表达。结果：各组ERK1／2蛋白表达无显著性差异,大、小剂量人参三七组方组及倍他乐克组Ras.c-Raf-1、p-ERK1／2蛋白和Ras.c-Raf-1、ERK1／2mRNA表达均显著高于模型组(P＜0.05)。结论：人参三七组方能够促进缺血心肌Ras信号通路上的Ras.c-Raf-1及ERK1／2表达,说明人参三七组方可能通过Ras信号通路来促进血管新生。二、临床文献系统评价：对1994年-2009年在科技期刊上公开发表的益气活血中成药治疗不稳定心绞痛的临床文献进行系统评价。目的：对益气活血中成药治疗不稳定心绞痛临床研究进行系统评价。方法：①以中文标题：不稳定性心绞痛and(全部检索条目：中医or中药or中西医结合)and(全部检索条目：益气or活血)的检索式检索中国生物医学文献光盘数据库,年限为1994年1月至2009年12月。②由2位研究者按照文献纳入、排除标准独立进行文献筛选,意见不同者通过讨论解决。③获取文献的相关研究特征资料,并加以评价。④对合格的随机对照试验,进行定量的Meta分析。结果：①共检索到170个文献。②经两次筛选,剔除不符合要求以及重复报道的文献,最终共有17篇文献入选评价。③对17篇文献进行Meta分析,总病例数1644例。④益气活血中成药包括愈心痛胶囊、通心络胶囊、复方丹参滴丸(片)、舒心益脉胶囊、步长稳心颗粒、活血通脉胶囊、血府逐瘀胶囊。Meta分析显示,加用益气活血中成药干预对不稳定心绞痛患者心绞痛症状和心电图的改善较单纯西医基础治疗明显提高,能减少硝酸甘油的用量,显著降低血浆内皮素(ET)、血浆内α-颗粒膜蛋白(GMP-140)等与动脉粥样硬化斑块稳定相关指标。结论：益气活血中成药干预不稳定心绞痛在改善心绞痛的症状、心电图表现,减少硝酸甘油的用量以及降低ET、GMP-140方面有一定的疗效。但由于纳入研究的质量不高,还有待进一步研究。三、文献综述部分通过对123篇文献的梳理和总结,探讨和综述了VEGF、CD105与血管新生的关系及其研究进展,并对益气活血法促冠心病血管新生的研究文献进行了初步总结,提出今后应临床研究与体内外实验研究相结合,从多途径、多环节探讨中药促进冠心病缺血心肌血管新生的作用及信号转导机制。本研究以Ras蛋白信号通路为主线,观察人参、三七组方对血管生成过程中Ras信号转导系统的干预作用及其机制,属于中医药防治冠心病研究领域的原创性研究工作；借鉴CD105检测肿瘤微血管密度的方法,采用CD105检测缺血心肌的微血管密度,是检测指标的创新应用。更多还原

【Abstract】 Therapeutic angiogenesis in ischemic diseases has become a hot topic. The Chinese medicine of Tonifying Qi and Activating Blood has a long history and certain efficacy in treating coronary heart disease(CHD). It can improve myocardial ischemic and angiogenesis of myocardial ischemia, and it has been confirmed by a lot of clinical and experimental studies, suggesting that promoting angiogenesis may be one of important mechanisms of Chinese medicine in the treatment of CHD.This study is to probe the molecular mechanism that the Chinese medicine of Tonifying Qi and Activating Blood to promote angiogenesis in myocardial ischemia rats,to reveal the role of intervention in angiogenesis and molecular targets and provide experimental evidence for the prevention and treatment of ischemic disease.The research includes three parts:experimental research, systematic review and literature review.1. Experimental research This part includes 3 experiments:The 1st experiment:Stimulating angiogenesis effect of the Chinese medicine of Tonifying Qi and Activating Blood on the hearts of myocardial infarction rats.Objective:To study the stimulating angiogenesis effect of the Chinese medicine of Tonifying Qi and Activating Blood of Radix Ginseng and Radix Notoginseng formula on experimental myocardial infarction rats. Methods:A total of 100 Wistar rats were divided into normal control group, sham-operated group, untreated group, metoprolol (Betaloc) group, high-and low-dose Radix Ginseng and Radix Notoginseng formula groups. Model of myocardial ischemia was induced in the untreated group, the Betaloc group, the high-and low-dose Radix Ginseng and Radix Notoginseng formula groups by left anterior descending coronary artery ligation. After 12-day treatment, morphological changes of Myocardial cells were observed, serum VEGF volum was detected,microvessel density (MVD) in ischemic myocardium was detected by immunohistochemical method.Result:Radix Ginseng and Radix Notoginseng formula could reduce the myocardial cell degeneration and inflammatory cells,increase blood vessel density and serum VEGF Concentration. Conclusions:Radix Ginseng and Radix Notoginseng formula could protect ischemic myocardium, and had the roles of stimulating angiogenesis in ischemic myocardium rats after acute myocardial infarction and increasing the microvessel density in ischemic myocardium.The 2nd Experiment:Effects of the Chinese medicine of Tonifying Qi and Activating Blood on the genes expression of vascular endothelial growth factor receptor-2 and hypoxia-inducible factor-la of ischemic myocardium of rats.Objective:To investigate the effects of the Chinese medicine of Tonifying Qi and Activating Blood of Radix Ginseng and Radix Notoginseng formula on expressions of vascular endothelial growth factor receptor-2 (VEGFR-2) and hypoxia-inducible factor-la (HIF-la) in ischemic myocardium in rats with acute myocardial infarction. Methods:The methods of modeling and treating were same to the 1st experiment. After the administration, expressions of VEGFR-2 and HIF-1αproteins were detected by Western blotting, and expressions of VEGFR-2 and HIF-1αmRNAs were detected by real-time fluorescent quantitative polymerase chain reaction. Results:VEGFR-2 and HIF-la protein and mRNA expressions in the high-and low-dose Radix Ginseng and Radix Notoginseng formula groups and Betaloc were higher than those in the untreated group (P<0.05). There was a significant difference between the high-and low-dose Radix Ginseng and Radix Notoginseng formula groups (P<0.05). VEGFR-2 and HIF-1αprotein and mRNA expressions in Betaloc group were higher than the high-and low-dose Radix Ginseng and Radix Notoginseng formula groups(P<0.01). Conclusion:Radix Ginseng and Radix Notoginseng formula can up-regulate the protein and mRNA expressions of VEGFR-2 and HIF-la to improve myocardial ischemia so as to promote the development of collateral circulation in dose-dependent manner.The 3rd Experiment:Effects of the Chinese medicine of Tonifying Qi and Activating Blood on the downstream Ras signal transduction pathwayObjective:To investigate the effects of the Chinese medicine of Tonifying Qi and Activating Blood of Radix Ginseng and Radix Notoginseng formula on the downstream Ras signal transduction pathway and probe the the signal transduction pathway that the Chinese medicine of Tonifying Qi and Activating Blood promoted angiogenesis. Methods:The methods of modeling and treating were same to the 1st experiment. After the administration, expressions of Ras、c-Raf-1、ERK1/2 and p-ERK1/2 proteins were detected by Western blotting, and expressions of Ras、c-Raf-1、ERK1 and ERK2 mRNAs were detected by real-time fluorescent quantitative polymerase chain reaction. Results:ERK1/2 protein expression was not significantly different in different groups.Ras、c-Raf-1、p-ERK1/2 proteins and Ras、c-Raf-1、ERK1/2 mRNAs expressions in the high-and low-dose Radix Ginseng and Radix Notoginseng formula groups and Betaloc were higher than those in the untreated group (P<0.05). Conclusion:Radix Ginseng and Radix Notoginseng formula could up-regulate the protein expressions of Ras、c-Raf-1、p-ERK1/2and Ras、c-Raf-1、ERK1/2mRNA. It suggested Radix Ginseng and Radix Notoginseng formula possibly promote angiogenesis through Ras signal transduction pathway.2. System evaluation of clinical literature the Chinese patent medicine of Tonifying Qi and Activating Blood for Unstable Angina Pectoris:a systematic reviewObjective:To systematically analyze the effect of the Chinese patent medicine of Tonifying Qi and Activating Blood on unstable angina pectoris. Methods:①Search items were [Title:unstable angina pectoris] AND [All field:(Chinese medicine) OR (Chinese herb) OR (Integrated Traditional and Western Medicine)] AND [All field:(Tonifying Qi) OR (Activating Blood)], the search was applied to the CBM database (1994.1～2009.12).②Literature bolting was performed by 2 reviewers independently according to the standard of in and out, different opinions should be solved through negotiation.③The relation data of literature were extracted and evaluated.④A meta analysis was performed based on qualified literatures. Result:①170 literatures were found out.②Through twice blotting, there were 17 clinical study literatures that meeted the evaluate procedure.③17 literatures came into meta analysis procedures, the total number of cases were only 1644 cases.④The Chinese patent medicine of Tonifying Qi and Activating Blood concluded yu xin tong capsules, tong xin luo capsules, compound dan sen drop(tablet),shu xin yi mai capsules, bu chang wen xin granules,huo xue tong mai capsules,xue fu zhu yu capsules. Meta analysis showed that compared with the single conventional western medicine, additional use of Chinese patent medicine of Tonifying Qi and Activating Blood could significantly improve the symptoms of angina, the electrocardiogram, reduce the using amount of nitroglycerin and decrease plasma endothelin (ET), plasma a-granule membrane protein (GMP-140) which were close to the stability of atherosclerotic plaque.Conclusion:Meta analysis shows that Chinese patent medicine of Tonifying Qi and Activating Blood has some benefits in improving the symptoms of unstable angina, electrocardiogram and decreasing the using amount of nitroglycerin,ET and GMP-140. Due to lower quality of studies, the further study is need.3. Literature review123 literatures were read, collected and summarized, and progress on studies of the relationship among VEGF、CD105 and angiogenesis was explored and reviewed.A preliminary summary was conducted on literatures about Chinese medicine of Tonifying Qi and Activating Blood stimulating angiogenesis of coronary heart disease. We put forward that clinical studies combined with researches in Vitro and Vivo be conducted through many ways, many links to explore the role and signal transduction mechanisms through which chinese medicine stimulates angiogenesis of myocardial ischemia of coronary heart disease.The research choosed the Ras protein signaling pathway as the main line, and observed the intervention of Radix Ginseng and Radix Notoginseng formula on angiogenesis in Ras signal transduction system in rats and its mechanism.It is a original research on the field of Chinese medicine’s prevention and treatment of coronary heart disease.Borrowing CD105 method of detection of tumor micro vessel density, the research adopted CD105 to detect micro vessel density of myocardial ischemia.It is a innovative application of the examination index.更多还原