【作者】 王晓静；

【导师】 梁高林；

【作者基本信息】 中国科学技术大学 ， 分析化学， 2014， 博士

【摘要】 荧光分子探针分析技术是一种重要的分子检测技术,具有灵敏度高、选择性好、检测方便、检测限低等特点。近几十年来,荧光分子探针技术在环境科学、医药以及生命科学等领域被广为应用。在论文中我们发展了一种新型的荧光可控探针Ac-SAACQ-Gly-Gly-Gly-Lys (FITC),该探针能被用于连续的选择性在体外和活细胞中检测Cu2+和L-组氨酸。分子探针结构中包含的Gly-Gly-Gly-Lys能够增加探针的水溶性和细胞渗透性；分子探针结构中SAACQ部分能够与Cu2+发生螯合作用；分子探针的荧光信号则来自于异硫氰酸荧光素(FITC),当探针分子的SAACQ部分与Cu2+结合以后由于PET现象的出现导致荧光素的荧光发生淬灭。最终我们成功实现了体外和活细胞内Cu2+以及L-组氨酸的连续性检测。定点标记蛋白在检测蛋白的表达、组合、转录,理解细胞内部随时间空间的变化具有重要的意义。在过去的十年中荧光蛋白已经广泛的应用到标记相应的fusion蛋白,并且具有很好的分辨率。但是它们具有高分子量以及对细胞环境迟钝的缺点。而化学探针则具有高标记的特点。在论文中我们通过合理改进传统的基于1,2-氨基硫醇和2-氰基苯并噻唑(CBT)的氰基的缩合反应中的底物,将该反应应用到分子成像。我们的研究体系结合了巯基与马来酰亚胺之间的亲核加成反应和CBT的-CN与半胱氨酸(Cys)的N端之间的缩合反应,最后成功的对鸡蛋膜上蛋白质的Cys残基的进行荧光标记。在临床上癌症多药耐药(MDR)的存在是癌症病人实施化学治疗失败的主要原因。多药耐药不仅对接触肿瘤细胞的药物产生耐药,而且对一些未曾接触、与之化学结构和作用机制完全不同的药物也产生交叉耐药,因此这给癌症的治疗带来了很大的障碍。而纳米载药体系的出现为克服MDR带来重大的突破。在论文中我们合理的设计了四种抗癌药物1,2,3以及对照化合物3-Scr。其中,2是化疗药物阿霉素(DOX)的衍生物。其他化合物则是紫杉醇(taxol)的衍生物。1,2,3都包含一段Arg-Val-Arg-Arg (RVRR)的肽链,该肽链能够被furin酶特异性的识别和剪切。另外,RVRR肽链片段使底物具有很好的水溶性以及细胞渗透性。当药物分子被furin酶剪切或者还原剂还原以后,缩合反应就会发生形成多聚体,进而发生自组装形成纳米颗粒,同时增加细胞内化疗药物的有效浓度。更多还原

【Abstract】 Fluorescent probe analysis technology has been widely noted because of its high sensitivity, good selectivity, detection convenient, and low detection limit microanalysis techniques. In recent decades, the fluorescent molecular probe technology in environmental science, medicine and life sciences analysis and detection in large numbers and rapid development.In this dissertation, we developed a new fluorescence probe Ac-SAACQ-Gly-Gly-Gly-Lys (FITC) for sequentially and selectively sensing Cu2+and L-histidine (L-His) in vitro and in living cells for the first time. The new probe has good water-solublility and cell-permeablility because of the existence of Gly-Gly-Gly-Lys. The SAACQ motif used for Cu2+chelation clawer and the fluorescein isothiocyanate (FITC) used as fluorescence resource in the probe. The fluorenscence of probes can be quenched via PET mechanism after Cu2+chelation. Finally, we sucessfully achieved the sequentially and selectively sensing Cu2+and L-His in vitro and in living cells.Site-specific labeling of proteins is an important approach for direct visualization of protein expression, association, and translocation, understanding the spatial and temporal underpinnings of life inside cells. In the past decade, genetically encoded fluorescent proteins (FPs) have been widely used to label their fusion proteins with absolute specificity and spatial resolution. However, they have intrinsic shortcomings such as high molecular weight and insensitivity to cellular environment. The chemical probes for visualization of the proteins had been developed to improve the labeling efficiency.In this dissertation, we made the advantage of condensation reaction between the1,2-aminothiol group of cysteine (Cys) and the cyano group of2-cyanobenzothiazole (CBT), and finally applied the condensation reaction to molecular imaging by optimalizing the substrate. In our experiment, we realized the labeling of Cys residues on proteins of chicken eggshell membrane (ESM) by combining these two biocompatible reactions (nucleophilic addition between thiol and maleimide, and condensation between CBT and N-terminal Cys).Multidrug resistance (MDR) is the major factor in the failure of large forms of chemotherapy in cancer patients. The MDR induced the tumor cells become more and more refractory to not only its own target drugs, but also some unrelated drugs that with different chemical structures or functionalized mechanisms. Nowadays, MRD becomes a big abstacle for the cancer treatment. The development of nano-drug delivery systems made significant breakthrough for overcoming the MRD.In this dissertation, we rationally designed four anticancer drugs1,2,3and its scramble probe3-Scr.2is a doxorubicin (DOX) derivative chemotherapeutic drug. And the other are taxol derivative chemotherapeutic drugs.1,2, and3contained a Arg-Val-Arg-Arg (RVRR) peptides sequence which can be specifically recognized and cleaved by furin. And RVRR sequence has a good water-solubility and cell-permeable. After furin cleavaged and reduction, the condensation reaction would be happened. The intracellular self-assembly of nanoparticals were formed and enhanced the concentration of chemotherapeutic drugs.更多还原