基于DNAzyme的多功能纳米载体的构建及其在乳腺癌基因治疗中的应用

发布时间：2018-03-11 14:06

本文选题：乳腺癌　切入点：DNAzyme　出处：《河北大学》2017年博士论文　论文类型：学位论文

【摘要】：乳腺癌是危害女性健康的主要恶性肿瘤,它的发生、发展涉及多种基因的改变。随着分子生物学技术的迅猛发展,越来越多的乳腺癌相关基因被确认,基因治疗成为乳腺癌治疗的一个新途径和研究热点,近年来已被证明有着极大的临床应用前景。在众多基因疗法策略中,DNAzyme具有高效的催化活性及特异的序列识别能力,能催化目标mRNA特定部位的切割,同时因其具有廉价低毒、易于合成和修饰、高稳定性和特异性的优势倍受青睐。借助DNAzyme,我们可以靶向沉默肿瘤细胞中有助于癌症恶化的基因,从而特异性的抑制肿瘤的生长,实现个体化治疗。然而,如何实现DNAzyme安全有效的体内递送及如何高效实现其靶向基因沉默功能是DNAzyme作为基因治疗药物走向临床应用的瓶颈。为此,本论文设计了基于DNAzyme的可降解的、多功能的纳米花及DNAzyme“纳米海绵”,用于乳腺癌靶向基因治疗。具体工作主要分为以下两部分:1.针对乳腺癌的基因治疗中,使用单一基因沉默试剂疗效局限性及以往基因载体不易降解的问题,我们构建了一种可降解的、多功能的DNAzyme纳米花样结构(DNFs)。在构建的体系中,利用滚环复制放大技术引入了核酸适配体aptamer、沉默EGR-1和survivin双重基因的DNAzyme,通过靶向、双重基因沉默功能用于乳腺癌的基因治疗,同时合成DNA纳米花还可以作为药物载体,实现药物靶向运输。体外和体内的研究结果表明,DNFs具有高效的基因沉默功能,降低靶基因在mRNA和蛋白的表达水平,从而诱导细胞凋亡,而且相对于单一基因沉默功能的DNFs具有更强的抗肿瘤活性。2.针对乳腺癌光热治疗中产生的热抗性而降低治疗效果的问题,我们构建了一个基于DNAzyme沉默热休克蛋白(HSP)基因的“纳米海绵”,通过阳离子聚合物和一条具有多拷贝的单链DNAzyme简单装配而成,借助静电作用力吸附小分子光热试剂ICG。该体系在近红外光照射下,释放的DNAzyme通过基因沉默的方式,下调癌细胞的耐热基因HSP70,有效增强了肿瘤细胞对光热的敏感性,同时利用体系的光热效应使得肿瘤区域迅速升温,从而有效杀死肿瘤。体外研究表明,这个nanosponge-ICG治疗体系在光热治疗中可以下调HSP70蛋白的表达到正常水平,抑制热休克反应,提高治疗效率。同时,利用荧光、光声成像示踪到nanosponge-ICG可以有效富集在肿瘤组织中。体内抗肿瘤实验结果表明,nanosponge-ICG可以高效的抑制肿瘤生长,而且无明显的脏器及血液毒性,因此可以作为一种新型生物相容好的治疗试剂用于乳腺癌的光热、基因联合治疗。总之,本论文是以DNAzyme为基因沉默试剂,针对乳腺癌基因治疗,DNAzyme在递送过程中如何克服生理屏障及进入体内、细胞后,如何保证其安全、高效发挥催化活性,构建了两种纳米载体,并在体内外的抗肿瘤实验中显示高效的抗肿瘤活性。本文的研究结果为今后DNAzyme作为基因药物走向临床应用提供了基础性研究数据,无疑具有重要的科学意义。
[Abstract]:Breast cancer is a major malignant tumor affecting the health of women, its occurrence, development involves a variety of genetic changes. With the rapid development of molecular biology, breast cancer related genes are increasingly being recognised, gene therapy has become a new way for the treatment of breast cancer and the research hotspot in recent years has been shown to have great the clinical application of gene therapy. Among the strategies, sequence recognition ability of DNAzyme with catalytic activity and specificity of efficient cutting can catalyze the target mRNA specific sites at the same time, because of its cheap, low toxicity, easy synthesis and modification, high stability and specificity advantages favored. With the help of DNAzyme, we can target the silence the tumor cells contribute to the deterioration of the cancer gene, thus inhibiting tumor growth specifically to achieve individualized treatment. However, how to realize the safe and effective delivery and in vivo DNAzyme How to realize the targeted gene silencing function is the bottleneck of DNAzyme as a gene therapy drug to clinical application. Therefore, this paper designed a DNAzyme based biodegradable, and DNAzyme nano flower "multifunctional nano sponge" for breast cancer targeting gene therapy. The specific work is divided into the following two parts: 1. for breast cancer gene therapy, gene silencing effect using a single reagent and limitations of previous gene vector is not easy to degrade problem, we construct a biodegradable, DNAzyme nano pattern structure function (DNFs). In the construction of the system, rolling circle amplification technology into the aptamer aptamer the silencing of EGR-1 and Survivin double gene DNAzyme by targeting dual gene silencing function for gene therapy of breast cancer, while the synthesis of DNA nano flowers can also be used as a drug carrier for drug Targeting transport. In vitro and in vivo studies showed that DNFs is an efficient gene silencing function, reduce the target gene expression in mRNA and protein levels, thereby inducing apoptosis, and relative to the heat resistance of single gene silencing function DNFs has antitumor activity of.2. produced stronger photothermal therapy for breast cancer and reduce the treatment of the effect of the problem, we construct a silent heat shock protein based on DNAzyme (HSP) gene "nano sponge" by cationic polymer and a simple assembly with multiple copies of a single stranded DNAzyme, with the help of electrostatic adsorption of small molecules in the system of photothermal reagent ICG. near infrared light irradiation, the release of DNAzyme by gene the silent way, heat resistant gene HSP70 was down regulated in cancer cells, effectively enhance the sensitivity of tumor cells to heat, while using the photothermal effect of tumor system Regional rapid warming, so as to effectively kill the tumor. In vitro studies showed that the expression of nanosponge-ICG HSP70 protein in the treatment system can be reduced to the normal level in photothermal therapy, inhibition of the heat shock response, improve the efficiency of treatment. At the same time, using fluorescence and photoacoustic imaging tracer to nanosponge-ICG can effectively enriched in tumor tissue in vivo anti-tumor experiments. Show that nanosponge-ICG can inhibit tumor growth and high efficiency, no obvious toxicity in organs and blood, it can be treated as a new reagent good biocompatibility for photothermal breast cancer, gene therapy. In conclusion, this paper is based on the DNAzyme gene silencing reagents for gene therapy of breast cancer, DNAzyme in the delivery process how to overcome the physiological barrier and into the body cells, how to ensure the safety and efficient catalytic activity, and constructs two kinds of nanoparticles, It also shows high anti-tumor activity in vivo and in vitro. The results of this study provide a basic scientific data for the future application of DNAzyme as a gene drug to clinical application.

【学位授予单位】：河北大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9;TB383.1

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