本文選題：乳腺癌　切入點：DNAzyme　出處：《河北大學》2017年博士論文　論文類型：學位論文

【摘要】：乳腺癌是危害女性健康的主要惡性腫瘤,它的發(fā)生、發(fā)展涉及多種基因的改變。隨著分子生物學技術的迅猛發(fā)展,越來越多的乳腺癌相關基因被確認,基因治療成為乳腺癌治療的一個新途徑和研究熱點,近年來已被證明有著極大的臨床應用前景。在眾多基因療法策略中,DNAzyme具有高效的催化活性及特異的序列識別能力,能催化目標mRNA特定部位的切割,同時因其具有廉價低毒、易于合成和修飾、高穩(wěn)定性和特異性的優(yōu)勢倍受青睞。借助DNAzyme,我們可以靶向沉默腫瘤細胞中有助于癌癥惡化的基因,從而特異性的抑制腫瘤的生長,實現(xiàn)個體化治療。然而,如何實現(xiàn)DNAzyme安全有效的體內遞送及如何高效實現(xiàn)其靶向基因沉默功能是DNAzyme作為基因治療藥物走向臨床應用的瓶頸。為此,本論文設計了基于DNAzyme的可降解的、多功能的納米花及DNAzyme“納米海綿”,用于乳腺癌靶向基因治療。具體工作主要分為以下兩部分:1.針對乳腺癌的基因治療中,使用單一基因沉默試劑療效局限性及以往基因載體不易降解的問題,我們構建了一種可降解的、多功能的DNAzyme納米花樣結構(DNFs)。在構建的體系中,利用滾環(huán)復制放大技術引入了核酸適配體aptamer、沉默EGR-1和survivin雙重基因的DNAzyme,通過靶向、雙重基因沉默功能用于乳腺癌的基因治療,同時合成DNA納米花還可以作為藥物載體,實現(xiàn)藥物靶向運輸。體外和體內的研究結果表明,DNFs具有高效的基因沉默功能,降低靶基因在mRNA和蛋白的表達水平,從而誘導細胞凋亡,而且相對于單一基因沉默功能的DNFs具有更強的抗腫瘤活性。2.針對乳腺癌光熱治療中產生的熱抗性而降低治療效果的問題,我們構建了一個基于DNAzyme沉默熱休克蛋白(HSP)基因的“納米海綿”,通過陽離子聚合物和一條具有多拷貝的單鏈DNAzyme簡單裝配而成,借助靜電作用力吸附小分子光熱試劑ICG。該體系在近紅外光照射下,釋放的DNAzyme通過基因沉默的方式,下調癌細胞的耐熱基因HSP70,有效增強了腫瘤細胞對光熱的敏感性,同時利用體系的光熱效應使得腫瘤區(qū)域迅速升溫,從而有效殺死腫瘤。體外研究表明,這個nanosponge-ICG治療體系在光熱治療中可以下調HSP70蛋白的表達到正常水平,抑制熱休克反應,提高治療效率。同時,利用熒光、光聲成像示蹤到nanosponge-ICG可以有效富集在腫瘤組織中。體內抗腫瘤實驗結果表明,nanosponge-ICG可以高效的抑制腫瘤生長,而且無明顯的臟器及血液毒性,因此可以作為一種新型生物相容好的治療試劑用于乳腺癌的光熱、基因聯(lián)合治療�？傊�,本論文是以DNAzyme為基因沉默試劑,針對乳腺癌基因治療,DNAzyme在遞送過程中如何克服生理屏障及進入體內、細胞后,如何保證其安全、高效發(fā)揮催化活性,構建了兩種納米載體,并在體內外的抗腫瘤實驗中顯示高效的抗腫瘤活性。本文的研究結果為今后DNAzyme作為基因藥物走向臨床應用提供了基礎性研究數(shù)據(jù),無疑具有重要的科學意義。
[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.

【學位授予單位】：河北大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R737.9;TB383.1

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