藻蓝蛋白—酞菁复合物的制备与性质研究

发布时间：2018-02-04 05:35

本文关键词： 光动力治疗藻蓝蛋白酞菁复合物　出处：《福州大学》2014年硕士论文　论文类型：学位论文

【摘要】：光动力疗法(Photodynamic Therapy,PDT)是近几年来迅速发展起来的一种选择性治疗增生性病变的新技术,已成为当前抗肿瘤研究领域的热点之一。光动力疗法的三要素是光、光敏剂和氧,其中光敏剂是关键因素。由于在近红外区(650nm-750nm)具有良好的光物理光化学性质,酞菁(Phthalocyanine,Pc)作为第二代光敏剂的应用前景已受到广泛关注。但由于酞菁大环共轭体系的强疏水作用,多数酞菁化合物水溶性较低,不利于其实际应用。另一方面,藻蓝蛋白(Phycocyanin,PC)是一种具有良好水溶性的卟啉类色素蛋白,已有研究表明,藻蓝蛋白具有光敏活性,作用光谱处于550nm-650nm,但光敏活性有待进一步提高。着眼于利用酞菁与藻蓝蛋白的各自优势,进而构建具有双功能基的复合光敏剂,本论文围绕藻蓝蛋白与酞菁复合物的制备、光谱性质、光动力活性开展研究,主要研究内容和结果总结如下:1.藻蓝蛋白的提取与纯化本文选择藻胆蛋白含量较高的螺旋藻作为提取原料。先采用反复冻融法从螺旋藻中得到藻蓝蛋白粗提液,再将粗提液经过(NH4)2S04盐析进行初步的提纯。盐析后的藻蓝蛋白通过DEAE Sepharose Fast Flow离子交换层析法和葡聚糖凝胶层析法进一步纯化。电泳和光谱测试表明所获得的藻蓝蛋白具有较高纯度,光谱纯度达到4.0以上,分子量为115kDa左右。2.酞菁的合成与表征合成和表征了三种酞菁,分别是1-[4-(2-氨基乙基)苯氧基]酞菁锌(简称ZnPcNH2),1,8(11),15(18),22(25)-四(3-羧基苯氧基)酞菁锌(简称(4),1-[4-(2-羧基乙基)苯氧基]酞菁锌(简称E1)。文献检索表明,ZnPcNH2酞菁配合物及其前驱体尚未见报道。3.藻蓝蛋白-酞菁复合物的制备本文通过非共价结合与共价结合两种方式来构建藻蓝蛋白-酞菁复合物。对于非共价结合途径,首先通过光谱法研究了一系列酞菁与藻蓝蛋白的相互作用。进而,选取了六种酞菁,通过温育交换法制备了六种非共价结合复合物。对于共价结合途径,选取了两种氨基衍生物及两种羧基衍生物修饰的酞菁锌,采用戊二醛连接法以及成酰胺反应,获得了四种共价复合物。同时,通过光谱法对复合物的组成进行了分析。4.藻蓝蛋白-酞菁复合物的性质研究研究了复合物在水溶液中电子吸收光谱、荧光光谱和存在状态。评价了部分酞菁-藻蓝蛋白复合物对人体肝癌细胞HepG2的光动力抑制活性。结果表明,无取代酞菁锌与藻蓝蛋白的复合物(ZnPc-PC,结合比为8:1)光动力抑制HepG2的IC50值为2.1μM,四羧基酞菁锌和藻蓝蛋白(C4-PC,结合比为6:1)光动力抑制HepG2的IC50值为0.93μM,上述两种复合物不但显示了显著的光动力抗癌活性,且活性明显高于藻蓝蛋白和相应的酞菁锌,值得对其深入开展研究。
[Abstract]:Photodynamic thermotherapy (PDT) is a new technique for selective treatment of proliferative lesions developed rapidly in recent years. The three elements of photodynamic therapy are light, Guang Min and oxygen. Guang Min is the key factor. Due to its good photophysical and photochemical properties in the near infrared region of 650nm-750nm, the phthalocyanine is a phthalocyanine. The application prospect of Pc) as the second generation Guang Min has been widely concerned. However, because of the strong hydrophobic effect of phthalocyanine macrocyclic conjugation system, most phthalocyanine compounds have low water solubility, which is not conducive to their practical application. Phycocyanin (PCP) is a porphyrin pigment protein with good water solubility. It has been studied that phycocyanin has Guang Min activity. The action spectrum was at 550 nm ~ 650 nm, but Guang Min activity needed to be further improved. The aim of this study was to make use of the respective advantages of phthalocyanine and phycocyanin, and then to construct a bifunctional complex Guang Min. In this paper, the preparation, spectral properties and photodynamic activity of phycocyanin and phthalocyanine complexes were studied. The main contents and results are summarized as follows:. 1. Extraction and purification of phycocyanin. In this paper, spirulina with high phycobilis content was selected as raw material. Firstly, the crude extract of phycocyanin was obtained from Spirulina platensis by repeated freeze-thaw method. Then the crude extract was passed through NH _ 4). 2S04 was purified by salting-out. Phycocyanin was purified by DEAE Sepharose Fast. Flow ion exchange chromatography and dextran gel chromatography were further purified. Electrophoresis and spectral analysis showed that the obtained phycocyanin had high purity. The spectral purity is over 4.0, and the molecular weight of phthalocyanine is about 115kDa. The synthesis and characterization of phthalocyanine are 1-. [Zinc phthalocyanine (ZnPcNH _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2H _ 2O _ 2). Zinc phthalocyanine. [Zinc phthalocyanine (E1). Preparation of phycocyanin-phthalocyanine complexes. In this paper, phycocyanin-phthalocyanine complexes were constructed by non-covalent binding and covalent binding. For non-covalent combination pathways. Firstly, the interaction of a series of phthalocyanines with phycocyanin was studied by spectral method. Then, six kinds of phthalocyanines were selected and six non-covalent complexes were prepared by the method of thermo-exchange. Two amino derivatives and two carboxyl derivatives modified zinc phthalocyanine were selected and four covalent complexes were obtained by glutaraldehyde bonding method and amide-forming reaction. The composition of phycocyanin-phthalocyanine complex was analyzed by spectroscopic method. The properties of phycocyanin-phthalocyanine complex were studied. The electronic absorption spectra of the complex in aqueous solution were studied. The photodynamic inhibitory activity of partial phthalocyanine-phycocyanin complex on human hepatoma cell line HepG2 was evaluated. The IC50 value of zinc phthalocyanine and phycocyanin complex, ZnPc-PCS, was 2.1 渭 M for photodynamic inhibition of HepG2. The IC50 value of the photodynamic inhibition of HepG2 by zinc tetracarboxylphthalocyanine and phycocyanin C4-PC4 with a binding ratio of 6: 1 was 0.93 渭 M. These two complexes not only showed remarkable photodynamic anticancer activity, but also had higher activity than phycocyanin and zinc phthalocyanine.
【学位授予单位】：福州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;O641.4

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