口蹄疫病毒样颗粒作为抗癌药物靶向载体的研究

发布时间：2019-07-01 10:37

【摘要】：病毒样颗粒(Virus-like particle VLPs)是一种无病毒基因组、不具有感染力的、空间构象与天然病毒极其相似的多蛋白结构。VLPs与天然病毒相似的免疫原性为新型候选疫苗的生产提供了可能,随着乙型肝炎病毒VLPs疫苗和人乳头瘤病毒VLPs疫苗相继上市,其他多种VLPs疫苗处于临床试验阶段或者临床前期研究阶段,这些疫苗研究方面的成功使VLPs成为应对紧急暴发的流行性疾病的新一代安全疫苗。到目前为止,已经有超过30个病毒科的100多个病毒属的病毒样颗粒在体外得到制备,并处于疫苗研究的不同阶段。除了在疫苗方面的应用外,VLPs独特的生物特性也使其成为优良的生物载体,VLPs作为纳米级材料,在生物载体方面具有许多独特的优势：由衣壳蛋白有机结合而形成的病毒样颗粒直径一般在20～150 nm之间,比表面大,承载能力强;组成病毒样颗粒的蛋白表面有许多靶细胞的结合位点,有利于靶向递送和结合；病毒样颗粒能够形成一定的稳定结构,如乙肝病毒、人乳头瘤病毒、MS2噬菌体、Qp噬菌体的二十面体结构,它们大多由单一外壳蛋白组装而成,能够经受较大范围内的pH值和温度的变化,稳定性较高；在VLPs表面展示出许多氨基酸残基,这些氨基酸残基可以和核酸、蛋白质、小分子药物上的一些基团通过化学键链接,形成稳定复合体,从而发挥病毒样颗粒在运载纳米材料、医学药物、外源基因方面的功能。抗癌药物的毒副作用是限制其发挥作用的重要原因,提高药物的靶向性,增加抗癌药物在肿瘤组织的积累,是目前抗癌药物的发展趋势之一。本研究为了提高抗癌药物进入肿瘤细胞的靶向性,借助大肠杆菌原核表达系统体外融合表达亚洲一型口蹄疫病毒的衣壳蛋白,并组装出口蹄疫病毒样颗粒,将抗癌药物化学偶联到口蹄疫病毒样颗粒的表面,利用口蹄疫病毒衣壳蛋白VP1表面的RGD保守肽段能够靶向识别整合素受体阳性细胞的特性,将抗癌药物运载到特定的癌细胞中,来达到靶向治疗的目的。研究结果显示,体外表征抗癌药物DOX与病毒样颗粒的复合物,发现其在中性和偏碱性的体系中较为稳定,而在偏酸性的体系中易于药物释放,这一特点可保证复合物在体内稳定运输,并在靶细胞的酸性环境中迅速释放。通过共聚焦显微镜技术和细胞凋亡检测发现,复合物和单独的抗癌药物相比,能够在靶细胞中大量积累,说明病毒样颗粒能够起到靶向运输抗癌药物的作用,细胞凋亡实验表明,复合物能够引起不同的癌细胞凋亡。初步在体外研究证明,口蹄疫病毒样颗粒可以作为一种潜在的抗癌药物载体。
[Abstract]:Virus-like particle VLPs is a non-viral genome that is not infectious, and the spatial conformation is very similar to that of the natural virus. The immunogenicity of VLPs with natural viruses provides a potential for the production of new candidate vaccines, and with the subsequent marketing of VLPs and human papillomavirus VLPs, other VLPs vaccines are in the clinical trial phase or in the preclinical phase of the study, The success of these vaccine studies has made VLPs a new generation of safe vaccines that deal with the epidemic's epidemic. To date, more than 100 virus-like particles of more than 30 viridae have been prepared in vitro and are in different stages of the vaccine study. In addition to the application in the field of vaccines, the unique biological characteristics of VLPs also make it an excellent biological carrier, VLPs as nanoscale materials, and have a number of unique advantages in the aspect of biological carriers: the diameter of the virus-like particles formed by the organic binding of the capsid proteins is generally between 20 and 150 nm, the surface of the protein which is composed of the virus-like particles has a binding site of a plurality of target cells, is favorable for targeting delivery and binding, and the virus-like particles can form a certain stable structure, such as the hepatitis B virus, the human papilloma virus and the MS2 bacteriophage, The icosahedral structure of the Qp bacteriophage, which is mostly assembled from a single shell protein, can be subjected to changes of pH value and temperature in a large range, and the stability is high; a plurality of amino acid residues are displayed on the surface of the VLPs, and the amino acid residues can be combined with the nucleic acid and the protein; Some of the groups on the small molecular medicine are linked through chemical bonds to form a stable complex, so that the function of the virus-like particles in carrying nano materials, medical drugs and foreign genes can be realized. The toxic and side effect of the anti-cancer medicine is an important reason for limiting the role of the anti-cancer drug, the targeting property of the medicine is improved, the accumulation of the anti-cancer drug in the tumor tissue is increased, and the anti-cancer drug is one of the current development trends of the anti-cancer drugs. In order to improve the targeting property of the anti-cancer drug into the tumor cell, the capsid protein of the Asia-type foot-and-mouth disease virus is fused in vitro by the E. coli prokaryotic expression system, and the foot-and-mouth disease virus-like particle is assembled, and the anti-cancer drug is chemically coupled to the surface of the foot-and-mouth disease virus-like particle, The RGD conserved peptide segment of the VP1 surface of the foot-and-mouth disease virus capsid protein can be used for targeting the characteristic of the integrin receptor positive cell, and carrying the anti-cancer drug into a specific cancer cell to achieve the aim of targeted therapy. The results showed that the compound of DOX and the virus-like particles of the anti-cancer drug was characterized in vitro, and it was found to be more stable in the system of neutral and partial alkaline, and it was easy to release the drug in the acid-biased system, which can ensure the stable transportation of the complex in the body. And is rapidly released in the acidic environment of the target cell. by means of confocal microscopy and cell apoptosis detection, the complex and the individual anti-cancer drugs can accumulate in a large amount in the target cell, The complex can cause apoptosis of different cancer cells. In vitro studies have shown that foot-and-mouth disease virus-like particles can be used as a potential anti-cancer drug carrier.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79

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