【作者】 余磊；

【导师】 韩雅莉；

【作者基本信息】 广东工业大学 ， 应用化学， 2012， 博士

【摘要】 随着人口结构的老龄化和人民生活水平的提高,心脑血管疾病已成为威胁人类健康的一大杀手,心肌梗塞、脑中风等血栓性疾病的发病率正逐年提高。溶栓是治疗血栓性疾病有效的手段,但目前临床使用的溶栓药物有异常出血等副作用,而且价格昂贵,因此迫切需要研制高效、快速、副作用小,价廉的新型溶栓药物。本文从两种昆虫(地鳖、黄粉虫)体内提取了一类纤溶酶,研究了其溶栓作用、抗血管生成作用和抑制肿瘤作用等性质。并对这两种昆虫的纤溶酶氨基酸序列进行了分析,利用生物信息学方法模拟了它们的三维结构,找到了这两种酶的活性中心。设计并合成一种新型高分子化合物,模拟该类纤溶酶。为深入研究该类纤溶酶的药理性质,开发廉价的新型溶栓药物奠定了理论基础。本文利用离子交换层析技术和凝胶层析技术,从黄粉虫和地鳖体内分离纯化了两种纤溶酶,分别命名为黄粉虫纤溶酶(YFP)和地鳖纤溶酶(EFP)。利用硫酸蒽酮法,测量了这两种纤溶酶的含糖量；利用SDS-聚丙烯酰胺凝胶电泳测量了这两种纤溶酶的相对分子量；纤维蛋白平板实验证明这两种酶都具有纤溶活性,并研究了金属离子、EDTA、尿素、巯基乙醇对这两种酶活性的抑制作用,以及他们最适宜的温度和pH值。用红外光谱表征了两种纤溶酶；鸡胚尿囊膜实验证明这两种纤溶酶都能抑制血管生成；荷瘤小鼠动物实验证明这两种纤溶酶都具有抑制肿瘤细胞的效果。研究结果表明：黄粉虫纤溶酶相对分子质量约为56.1kD,糖含量为7.084%,Na+和K+对黄粉虫纤溶酶的纤溶活力基本无影响,而Mg2+和Ca2+则对其活力有一定的抑制作用,EDTA、尿素和巯基乙醇也能抑制其纤溶活性。黄粉虫纤溶酶的最适宜反应温度为25℃-45℃,最适宜pH值为7.5。做酶促反应动力学实验检测了黄粉虫纤溶酶对不同底物的Km值,得出结论：该酶跟不同的底物结合能力是不一样的。跟酯键结合能力较强,跟酰胺键结合能力次之,跟酪蛋白肽键结合能力最弱。地鳖纤溶酶相对分子质量约为41.3kD,糖含量为10.05%。Na+与K+对地鳖纤溶酶活性几乎没有影响,Mg2+,Ca2+对其活性有一定的抑制作用,EDTA、尿素和巯基乙醇也能抑制其纤溶活性。地鳖纤溶酶的最适宜反应温度为40℃,最适宜pH为8.0。利用caspase-3活性检测试剂盒,检测了地鳖纤溶酶诱导H22和S180肿瘤细胞凋亡时caspase-3的活性,得出结论,地鳖纤溶酶体内诱导的S180和H22肿瘤细胞凋亡的其中一个通路是caspase-3活化机制。利用生物信息学方法,对黄粉虫纤溶酶和地鳖纤溶酶进行了结构分析,用biosun软件的同源模建技术,模拟了它们的三维结构。利用goldkey软件,对这两种酶的氨基酸序列进行了分析,重点讨论了其柔性、亲水性、等电点跟催化活性之间的关系。找到了它们的活性中心和底物结合部位。这两种昆虫纤溶酶的活性中心都是组氨酸、丝氨酸和天冬氨酸三个氨基酸残基,都位于球蛋白中心凹穴处,底物结合部位都是丝氨酸、天冬氨酸和甘氨酸。从微观分子水平上阐述了该类酶水解纤维蛋白的机理是催化精氨酸-赖氨酸之间的肽键水解,与报道的纤维蛋白的溶解机理相符,为模拟酶的合成提供了思路。比较这两种昆虫纤溶酶,发现他们的同源性很高,三维结构类似,柔性相近,亲水性和等电点有一定的差异。纤溶酶活性中心是组氨酸、丝氨酸和天冬氨酸三个氨基酸残基,其侧链的官能团分别是：咪唑基、羟基和羧基。因此设计乙烯基咪唑、丙烯醇和丙烯酸三元共聚,合成一种新型高分子共聚物(模拟酶)。该共聚物在国内外均未见报道,其主链为碳链,侧链布满了咪唑基、羟基和羧基。以渗透压法,测量了模拟酶的数均分子量；以纤维蛋白平板实验中纤溶圈的面积,表征模拟酶的活性,得到模拟酶的最佳合成条件；研究了金属离子和pH值对模拟酶纤溶活性的影响。凝胶色谱实验,得到模拟酶的平均分子量,以及分子量分布；利用不同环境下模拟酶的粘度检测,对其微观结构作了初步探讨。得出结论,该模拟酶的数均分子量为：4.7×104；最佳反应条件为：乙烯基咪唑、丙烯酸、丙烯醇按投料比1：20：1；苯为溶剂；偶氮二异丁基腈为引发剂；反应温度为80℃。体外血块溶解实验证明,20%的模拟酶溶液能很好的在体外溶解血块,模拟酶浓度越高,溶栓能力越强。金属离子对模拟酶的纤溶活性没有显著性影响,pH为6和9时,模拟酶纤溶活性最高。并用红外、核磁共振表征了模拟酶的结构。更多还原

【Abstract】 With the development of economic and life level, cardiovascular disease, such as acute myocardial infarction and stroke, are one of the life-threatening diseases that affect more and more people each year. The thrombolysis is a main therapeutic approach to treat thrombosis disease. Despite its tremendous clinical success, several major limitations of the therapy still need to be addressed, including bleeding and high price. Thus, identifying new therapeutic targets and developing safe and efficient thrombolytic agents are the focal points in the field of thrombolysis. In this thesis, two kinds of insects’ plasmins were extracted from Tenibrio molitor (yellow mealworm beetle) and Eupolyphaga sinensis (ground beetle), and their properties of thrombolysis, anti-angiogenesis and tumor-suppression were studied. The amino acid sequences of the plasmins were analyzed, while their three-dimensional structures and active sites were determined by the method of bioinformatics. A novel of polymer (mimic enzyme) was designed and synthesized to simulate the plasmins, which would lay a necessary theory and practical foundation for the development of new thrombolytic drugs.Two kinds of insects’ plasmins were purified form yellow mealworm beetles and Eupolyphaga sinensis, by DEAE-32cellulose chromatography column and SephadexG-75dextran gel column, which named YFP(fibrinolytic protein form Yellow mealworm beetle) and EFP(fibrinolytic protein form Eupolyphaga sinensis). The fibrinolytic activity were detected by the fibrin plate method; their sugar content were detected by the anthrone-sulfuric acid method, and their molecular weight were estimated by SDS-PAGE. The chick embryo chorioallantoic membrane experiment proved that the two kinds of plasmins could inhibit angiogenesis, and both of them had anti-tumor effects, through animal experiment of tumor-bearing mice. The results showed that the relative molecular mass of YFP was about56.1kD; sugar content of YFP was7.084percent; The fibrinolytic activity of YFP had no influence on Na+and K+, while the Mg2+, Ca2+, EDTA, urea and mercaptoethanol has some inhibitory effect to its vitality; the optimal reaction pH value and temperature of YFP were pH7.5and25℃-45℃, respectively. The Km of YFP were tested by the experiments of enzymatic kinetic reaction, It could be concluded that the capability of the YFP with various substrates was in the follwing order:ester bond＞amide bond＞peptide bond. The relative molecular mass of EFP was about41.3kD; sugar content of EFP was10.05percent; The fibrinolytic activity of EFP had no influence on Na+and K+ while the Mg2+, Ca2+,EDTA, urea and mercaptoethanol has some inhibitory effect to its vitality:the optimal reaction pH value and temperature of EFP were pH8.0and40℃. respectively. The EFP had anti-tumor effects on SI80and H22solid tumors, and one pathway of apoptosis of S180and H22tumor cells induced by EFP was activation of caspase-3.using the bioinformatics methods, the3D-structures of EFP and YFP were simulated by the the "biosun" computer program, while the isoelectric point, hydrophilicity, and flexibility of the sequences were analyzed by the the "goldkey" computer program. The acive sites of the YFP and EFP both were Histidine, Serine and Aspartic acid, and in the same position, while the Substrate binding sites both were Serine, Aspartic acid and Glycine. the plasmins’ catalytic mechanism was the catalyzing hydrolysis of the peptide bonds between the Arginine and the Lysine.The acive sites of the insects’ plasmins were Histidine, Serine and Aspartic acid, and the functional groups of the three amino acids were Imidazole, hydroxyl and carboxyl groups. So a novel of polymer (mimic enzyme) was designed and synthesized through by copolymerization with raw materials of acrylic acid, allyl alcohol and vinyl imidazole. The molecular weight of the mimic enzyme were estimated by osmotic method, and The fibrinolytic activity of the mimic enzym were detected by the fibrin plate method.The optimum reaction conditions were vinyl imidazole. acrylic acid, allyl alcohol in the ratio of1:20:1, benzene as solvent. zobisisobutylnitrile as initiator, and80℃. In the optimum reaction conditions, the molecular weight of the mimic enzyme was4.7×104. As the concentrations increased, the fibrinolytic activity of the mimic enzyme was increasing, and blood clots could be dissolved in20%mimic enzyme solution. The fibrinolytic activity of the mimic enzyme had no significant influence by metal ions, and the optimal pH of the mimic enzyme were6and9. The polymer was characterized by IR and NMR, and it’s viscosity was studied also.更多还原