靶向型纳米基因载体递送小核酸miR-34a抑制肿瘤的增殖与迁移

发布时间：2018-03-12 09:27

本文选题：主动靶向　切入点：小核酸　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：在过去的数十年间,随着对癌症发展分子机制的不断探索,靶向型基因递送体系应运而生,其中一些纳米药物已经投入到临床使用中,其他很多纳米基因药物也已进入临床试验阶段。为了提高药物的安全性、可靠性,解决药物的溶解度低、对身体正常部位产生毒副作用、生物学不稳定、无法跨越生理屏障等问题,实现纳米体系的主动靶向性,减少网状内皮吞噬系统(RES)对纳米载体的捕获,使纳米体系可以通过肿瘤细胞表面的受体进行受体-配体相互作用而进入细胞,实现主动靶向治疗成为基因药物研究的重中之重。基于上述原因,本论文通过EDC/NHS法,将能够特异性识别癌胚抗原6的单域抗体修饰到高分子纳米材料PEN表面,构建一种具有肿瘤特异靶向性的高分子纳米载体PEN-Nb。该载体对miR-34a有良好的结合能力,同时可以通过肿瘤细胞表面的癌胚抗原6受体进行受体-配体相互作用而进入细胞,在提高生物学稳定性的同时提高转染效率。miR-34a在胞内充分发挥生物学效应,抑制了肿瘤细胞MCF-7的增殖,促进其产生凋亡,使细胞周期阻滞在G1~S期,同时也抑制了肿瘤细胞的迁移浸润。为了进一步研究主动靶向性载体在活体水平上的治疗效率,我们以BALB/c-nu裸鼠构建异种移植瘤肺癌模型,以天然多糖硫酸软骨素对PAMAM进行修饰合成CD44靶向性纳米载体CS-PAMAM,以此递送小核酸miR-34a,观察该纳米递送体系在动物水平上的靶向性及对肿瘤的治疗效果。CS-PAMAM在细胞水平上有良好的生物相容性及CD44靶向性,在动物实验结果中,以CS-PAMAM递送miR-34a能够有效抑制肿瘤体积的增长,通过组织切片观察可以发现CS-PAMAM/miR-34a组有明显的原位凋亡的产生,并且该纳米体系并没有对小鼠的其他脏器产生任何的毒副作用。药物体内分布实验证明CS-PAMAM能够使miR-34a最大程度的在肿瘤部位蓄积,使其充分发挥生物学功能。综上,这两种肿瘤靶向型纳米基因递送体系均能通与过肿瘤细胞表面的受体进行受体-配体相互作用实现主动靶向,从而实现小核酸miR-34a的高效递送,充分发挥小核酸miR-34a的生物学功能,在细胞水平和动物水平上抑制肿瘤的增殖与迁移。以上研究,为构建主动靶向型纳米基因递送体系提供了新的策略,证明了靶向型纳米基因递送体系在肿瘤治疗领域的巨大潜力,为未来的临床应用奠定了良好的基础。
[Abstract]:Over the past few decades, as the molecular mechanisms of cancer development have been explored, targeted gene delivery systems have emerged, and some of these nanopharmaceuticals have been put into clinical use. Many other nanocrystalline drugs have also entered the clinical trial stage. In order to improve the safety and reliability of drugs, solve the low solubility of drugs, have toxic side effects on normal parts of the body, biological instability, It is impossible to cross the physiological barrier to achieve the active targeting of nano-system and reduce the capture of nano-carrier by reticular endothelial phagocytosis system (RES). So that the nanoscale system can enter the cell through receptor ligand interaction on the surface of tumor cells and realize active targeting therapy has become the most important part of gene drug research. Based on the above reasons, the EDC/NHS method is adopted in this paper. A novel polymer nano-carrier PEN-Nb, which can specifically recognize carcinoembryonic antigen 6, was constructed by modifying it onto the surface of the polymer nano-material PEN. The carrier has good binding ability to miR-34a. At the same time, the receptor-ligand interaction can be carried out by carcinoembryonic antigen 6 receptor on the surface of tumor cells, which not only improves the biological stability but also increases the transfection efficiency. MiR-34a exerts the full biological effect in the cell. It inhibited the proliferation of MCF-7, promoted its apoptosis, blocked the cell cycle in G1S phase, and also inhibited the migration and infiltration of tumor cells. In order to further study the therapeutic efficiency of active targeting vector at the in vivo level, We used BALB/c-nu nude mice to construct xenotransplantation tumor lung cancer model. PAMAM was modified with natural polysaccharide chondroitin sulfate to synthesize CD44 targeting nano-carrier CS-PAMAM, which was used to deliver small nucleic acid miR-34a. The targeting of the nano-delivery system at the animal level and the therapeutic effect of CS-PAMAM on tumor were observed at the cell level. Good biocompatibility and CD44 targeting. In animal experiments, CS-PAMAM delivery of miR-34a could effectively inhibit the growth of tumor volume, and in situ apoptosis could be found in CS-PAMAM/miR-34a group by histological observation. And the nanoparticles did not produce any toxic side effects on other organs of mice. The drug distribution in vivo showed that CS-PAMAM can make miR-34a accumulate in tumor sites to the greatest extent, so that it can give full play to its biological function. Both of these two tumor targeting nano-gene delivery systems can actively target the receptor ligand interaction with receptors passing through the surface of tumor cells, thus realizing the efficient delivery of small nucleic acid miR-34a and giving full play to the biological functions of small nucleic acid miR-34a. Inhibition of tumor proliferation and migration at the cellular and animal levels provides a new strategy for the construction of active targeting nanogene delivery systems. It is proved that the targeting nano gene delivery system has great potential in the field of tumor therapy, which will lay a good foundation for clinical application in the future.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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