茶黄素对胶原引起的血小板功能的影响及其抗血栓作用研究

发布时间：2018-01-02 14:37

本文关键词：茶黄素对胶原引起的血小板功能的影响及其抗血栓作用研究　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：心血管疾病,包括中风、血栓性疾病、动脉粥样硬化等,一直以来是导致死亡的主要原因之一,血小板在心血管疾病的发展过程中扮演着重要的角色,因此抗血小板治疗成为预防和治疗心血管疾病的主要手段。然而目前临床使用的抗血小板药物,如阿司匹林,存在着出血、引起胃肠道不适等副作用,寻找天然活性来源的抗血小板物质成为预防和治疗心血管疾病研究的热点之一。红茶是世界上消费量最大的饮料之一,流行病学研究表明饮用红茶具有预防心血管疾病的作用,其临床研究结果多集中在降血脂,改善血液循环等领域,在对血小板活性的影响方面较少涉及。茶黄素作为红茶的特征品质成分,具有抗氧化、抗炎、抗癌等多种生物活性,在心血管健康方面也表现出保护作用。然而,显少有针对茶黄素单体在血小板活性影响以及抗血栓作用方面的研究,尤其在其作用机理的深入探讨方面研究甚少。本研究探索了红茶主要活性成分茶黄素对胶原引起血小板活性的影响以及其抗血栓作用。以茶黄素为研究对象,将从人体血液中分离出来洗涤血小板,建立不同的研究体系,利用血小板聚集仪、流式细胞仪、血栓素A2试剂盒、免疫印迹等技术进行系统性的分析,并结合体内实验,利用C57小鼠研究茶黄素的止血功能和对体内血栓形成的影响。研究结果表明,茶黄素可以明显抑制由胶原、ADP、U46619等多种刺激剂引起的人体血小板聚集,且呈现出剂量依赖效应。利用流式细胞仪进行单个血小板激活检测时发现,茶黄素对蛇毒引起的P-选择素表达和纤维蛋白原的结合有明显的抑制作用。茶黄素可以抑制血小板在纤维蛋白原上的铺展,由此证明了茶黄素对血小板从外到内的信号通路的抑制活性。免疫印迹实验表明,茶黄素抑制胶原引起的血小板内脾酪酸激酶(Syk)的磷酸化,由此推测,Syk是茶黄素抑制胶原引起的血小板活性的一个重要的作用靶点。通过应用Syk的抑制剂白杉醇(Piceatannol)进行对比和叠加实验,进一步证明了茶黄素对血小板的作用靶点是Syk,同时,Syk下游两个蛋白PLCγ2和Akt的磷酸化受到相应抑制,也在本实验中得以验证。小鼠体内活性研究中,利用断尾技术研究茶黄素的止血活性,利用三氯化铁诱导形成体内动脉血栓模型,并采用血小板的体内荧光标记技术,研究茶黄素的抗血栓活性。茶黄素组和对照组相比,使小鼠肠系膜动脉血管形成血栓性堵塞的时间明显延长,充分证明了茶黄素对血小板功能的抑制。本文明确了茶黄素的体内外抗血栓活性,首次探明茶黄素抑制血小板活化的作用靶点,即茶黄素能够通过抑制血小板活化通路中的Syk以及PLCγ2磷酸化进而抑制由胶原引起的人体血小板活化和聚集,并能够显著抑制小鼠体内血栓的形成。作为饮食来源的天然活性成分,茶黄素表现出良好的抑制血小板活化的作用和抗血栓能力,有力地诠释了长期饮用红茶对人体心血管健康的保护作用。
[Abstract]:Cardiovascular disease, including stroke, thrombotic disease, atherosclerosis, has been one of the leading causes of death, platelet plays an important role in the development of cardiovascular disease, so antiplatelet therapy has become the main means of prevention and treatment of cardiovascular disease. However, current clinical use of antiplatelet drugs, such as aspirin, there is bleeding that cause gastrointestinal discomfort and other side effects, looking for sources of natural active antiplatelet substances become one of the hot research of cardiovascular disease prevention and treatment. Black Tea is one of the world's largest consumption of beverages, epidemiological studies have shown that drinking Black Tea has the role of prevention of cardiovascular disease, the clinical research results focused on reducing blood lipid, improve blood in the field of circulation, influence on platelet activity less involved. As the characteristics of Black Tea theaflavins The quality of ingredients with antioxidant, anti-inflammatory, anti-cancer and other biological activity on cardiovascular health also showed protective effects. However, few in theaflavin monomers in platelet activity and antithrombotic effect research, especially in the aspects of its mechanism in-depth study. This study explored the little active ingredients of tea Black Tea caused flavin platelet activity and antithrombotic of collagen. With theaflavins as the research object, will be isolated from human blood platelet washing out, establish different research systems, using platelet aggregation analyzer, flow cytometry, thromboxane A2 kit, immunoblotting analysis system the combined with in vivo, using effect of hemostatic function of tea C57 mice and emodin on thrombosis in vivo. The results showed that theaflavins can significantly inhibit the Collagen, ADP, U46619 and other stimuli induced human platelet aggregation, and showed a dose dependent effect. The use of a single activated platelet flow cytometry showed that P- selection of theaflavin on venom from the expression and binding of fibrinogen has obvious inhibiting effect of theaflavins can inhibit platelet. Spreading on fibrinogen, which showed inhibitory activity of theaflavin on platelet signaling from outside to inside. Immunoblotting experiments showed that theaflavins inhibited collagen induced platelet in spleen tyrosine kinase (Syk) phosphorylation, indicating that Syk is an important target of platelet activity of theaflavins inhibition of collagen induced by Syk. The inhibitor paclitaxel (Piceatannol) were compared and the superposition experiment, further proved that the targets of theaflavins on platelet is Syk, at the same time, the downstream of Syk two Protein PLC gamma 2 and Akt phosphorylation by the corresponding inhibition, can be verified in this experiment. Study on the activity of mice, hemostatic activity of tea by using tail technology, in arterial thrombosis induced by ferric chloride, and the platelet in vivo fluorescence labeling technique, study the antithrombotic activity of theaflavins theaflavins. Group compared with the control group of mice mesenteric arterial thrombotic formation blocking time was prolonged, fully demonstrated the inhibition of theaflavins on platelet function. Antithrombotic activity Lear the theaflavins in vitro and in vivo, the target for the first time proven theaflavins inhibit platelet activation, namely theaflavin can pass inhibition of platelet activation pathway in Syk and PLC gamma 2 phosphorylation and inhibition of human platelet activation and aggregation induced by collagen, and can inhibit thrombosis in mice form As a natural active ingredient of dietary sources, theaflavins show good inhibition of platelet activation and antithrombotic ability, which strongly explain the long-term protection of black tea on human cardiovascular health.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R151

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