【作者】 李伟；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 博士

【摘要】 核酸适体是一类经体外人工进化程序筛选出的寡聚核苷酸。它能高效、特异地结合蛋白质等多种目标物质,且具有易合成、易功能化修饰、稳定性好等优点,在生物医学的基础研究和临床诊断与治疗等方面显示出巨大的应用前景。本文选择了与肿瘤发生、发展密切相关的人血管生成素为目标蛋白,基于核酸适体和荧光分析方法,以荧光核酸适体探针的设计及其在血管生成素分析中的应用为主线,开展了以下几方面工作:(1)设计合成核酸适体/核酸分子“光开关”复合物探针,发展了基于核酸适体和核酸分子“光开关”[Ru(bipy)2(dppz)]2+的血管生成素检测方法。血管生成素与核酸适体的结合引起核酸适体/核酸分子“光开关”复合物探针的荧光信号显著下降,从而实现血管生成素的检测,在2.5~20 nmol/L的浓度范围内有良好的线性关系,检测下限为1.5 nmol/L。该方法中探针设计简便,不需要直接对蛋白质及核酸适体进行荧光标记。(2)设计合成单荧光标记的核酸适体探针,发展了基于荧光各向异性技术的血管生成素检测方法。血管生成素与核酸适体的结合引起核酸适体探针的荧光各向异性信号显著增强,从而实现血管生成素的特异性检测,在2~55 nmol/L的浓度范围内有良好的线性关系,检测下限为1 nmol/L。利用该方法实现了肺癌病人血清中血管生成素的检测。(3)设计合成双荧光标记的核酸适体探针,发展了基于荧光共振能量转移的血管生成素检测方法。血管生成素与核酸适体的结合引起核酸适体的空间结构发生变化,使得核酸适体探针的荧光共振能量转移效率增加,从而实现血管生成素的高灵敏检测,在0.5~40 nmol/L的浓度范围内有良好的线性关系,检测下限为0.2 nmol/L。利用该方法实现了健康人和肺癌病人血清中血管生成素的检测。(4)设计合成两端芘标记的核酸适体探针,发展了基于激发态二聚体的血管生成素检测方法。血管生成素与核酸适体的结合引起核酸适体空间结构的变化,导致两端芘分子的靠拢而形成激发态二聚体,使得荧光信号发生显著变化,从而实现血管生成素的高特异、高灵敏检测,在0.2~30 nmol/L的浓度范围内有良好的线性关系,检测下限为0.1 nmol/L。(5)将核酸适体探针的应用从蛋白质的简便、快速、高特异、高灵敏的检测拓展到活细胞水平上的蛋白质实时分析。利用单荧光标记的核酸适体探针,结合激光共聚焦成像技术,对血管生成素的内化过程进行实时成像研究,发展了一种在活细胞水平上进行蛋白质分析的新方法。血管生成素-核酸适体复合物与细胞共培育,能选择性地结合到脐带静脉内皮细胞和乳腺癌细胞上;核染实验和Z-轴扫描结果证实,复合物能进入靶细胞,到达细胞核;实时成像结果证实,复合物能快速结合到靶细胞上并进入细胞;不同温度培养实验证实,复合物进入细胞是受培养温度影响的,提示为内吞作用方式。该方法采用核酸适体作为细胞水平上蛋白质识别和标记的工具,无需事先标记目标蛋白,无需对目标细胞预先进行基因操作,更不必将目标细胞进行固定,可实时反映细胞-蛋白相互作用的真实状况,有望用于跟踪和定位细胞代谢过程中分泌的细胞因子、生长因子、多肽、酶等重要生物分子。以上研究工作表明,基于荧光核酸适体探针的血管生成素分析方法无需使用抗体,操作简便,成本较低,均具有快速、灵敏、选择性高的特点,有望在血管生成素的相关基础研究和临床检验中得到应用。更多还原

【Abstract】 Aptamers are a novel class of synthetic DNA/RNA oligocleotides generated from in vitro selection to bind with proteins and various targets. Due to their high affinity and specificity, easy synthesis, conveniently functional modification and good stability, aptamers have shown great prospect for basic biomedical research and clinical diagnosis and therapy. In this dissertation, angiogenin, which is closely correlated with the occurrence and development of cancers, was chosen as the target protein. Aiming at the design of fluorescent aptamer probes and their applications to angiogenin analysis, a series of angiogenin assays based on aptamers and fluorescent analyses have been developed. The research work of this dissertation is summarized as follows:(1) Aptamer/nucleic acid“molecular light switch”complex probe was designed and synthesized, and a method for angiogenin detection based on aptamer and nucleic acid“molecular light switch”[Ru(bipy)2(dppz)]2+ was developed. This method took advantage of the obvious fluorescent signal decrease of aptamer/nucleic acid“molecular light switch”complex probe upon specific angiogenin/aptamer binding. By monitoring the change of fluorescent intensity, angiogenin detection could be achieved quickly. This assay could determine angiogenin in the range of 2.5~20 nmol/L with a detection limit of 1.5 nmol/L. In this assay, the design of probe is convenient since neither the aptamers nor target proteins should be labeled directly.(2) A singly fluorophore-labeled aptamer probe was designed and synthesized, and a method for angiogenin detection based on fluorescence anisotropy was developed. This method took advantage of the obvious fluorescence anisotropy signal increase of fluorophore-labelled aptamer probe upon specific angiogenin/aptamer binding. By monitoring the anisotropy change, angiogenin detection could be achieved specifically. This assay could determine angiogenin in the range of 2~55 nmol/L with a detection limit of 1 nmol/L. Angiogenin in serum samples from lung cancer patients have also been detected by using this method.(3) A dually fluorophore-labeled aptamer probe was designed and synthesized and a method for angiogenin detection based on fluorescence resonance energy transfer (FRET) was developed. This method took advantage of the FRET efficiency increase caused by configuration shift upon specific protein/aptamer binding. By monitoring the fluorescence intensity of donor and acceptor fluorophores and calculating the FRET efficiency, angiogenin detection could be achieved with high sensitivity. This assay could determine angiogenin in the range of 0.5~40 nmol/L with a detection limit of 0.2 nmol/L. Angiogenin in serum samples from health people and lung cancer patients have also been detected by using this method.(4) A dually pyrene-labeled aptamer probe was designed and synthesized and a method for angiogenin detection based on pyrene excimer was developed. The method took advantage of the obvious fluorescence signal change of excimer caused by configuration shift and encounter of the pyrene molecules at the both termini upon specific protein/aptamer binding. By monitoring the fluorescence intensity of aptamer probe, quantitative angiogenin detection could be achieved with high specificity and sensitivity. This assay could determine angiogenin in the range of 0.2~30 nmol/L with a detection limit of 0.1 nmol/L.(5) The applications of aptamer probes were extended from convenient, quick, highly sensitive and specific protein detection to real-time protein assay in live cells. A novel simple and quick method for visualizing the real-time cellular internalization process of angiogenin was developed by using singly fluorophore-labeled aptamer probe and confocal laser scanning microscopy. Specifically, when aptamer/angiogenin conjugates were added into cell cultures, conjugates could be selectively bound to human umbilical vein endothelial cells and human breast cancer MCF-7 cells. Nuclear staining and Z-axis scanning studies demonstrated that the aptamer/angiogenin conjugates were internalized to intracellular organelles of target cells, and dynamic confocal imaging studies indicated that the conjugates were quickly bound to target cells and internalized. Different temperature culture studies validated that the internalization of aptamer/angiogenin conjugates were dependent on culture temperature and implied an endocytosis process. This method provides the first evidence that a fluorophore-labeled aptamer could be used not only as a protein labeling agent but also as a protein recognition agent to report the actual interaction of proteins with cancer cells in real time without target protein prelabelling, target gene cloning and target cell fixing. This method also provides the promising potential of fluorophore-labeled aptamers used as recognition and labeling probes for tracking and localizing of some important biomolecules such as cell factors, growth factors, polypeptides and enzymes secreted by metabolic processes.The above fluorescent aptamer probe-based analytical methods are convenient and low-cost without using antibody. Angiogenin in serum and living cells could be analysed quickly, sensitively and selectively. Therefore, these aptamer-based methods have the potential to be used in basic research and clinical diagnosis concerning angiogenin.更多还原