【作者】 李敏；

【导师】 崔晓燕；

【作者基本信息】 华东师范大学 ， 无机化学， 2020， 硕士

【摘要】 生物体生理和病理的变化机制研究对预防和诊断疾病至关重要。细胞作为生物体基本的结构和功能单位,参与生物体内各种生理过程与变化。监测细胞内各种活性物质的种类、形成过程和细胞内微环境的变化,是研究生理和病理变化机制的基础。但是,由于细胞内分析物的浓度相对较低,因此迫切需要高灵敏度的新型成像工具。传统的成像方式,如计算机断层扫描,磁共振成像和超声检查等,缺乏足够的特异性和敏感性。正电子发射断层扫描灵敏度高,但是这种成像方式受限于严格的放射性化合物使用规定。而光学成像以其无创、实时和高分辨率成像为准确诊断疾病提供了希望。特别是利用小分子荧光技术进行的定位成像,实现了可视化细胞中的特定细胞器乃至整个活细胞,同时也成为了准确预防诊断疾病的有力工具。(1)利用细胞器物理性质监测脂滴的形成过程极性是生命体中的一个重要理化性质,在细胞生理过程的变化中发挥着重要作用。相比蛋白质、核酸和碳水化合物等,脂质结构的极性相对较小。而极性敏感型荧光探针分子可以很好地定位脂质物质。极性敏感型荧光探针在电子激发后,偶极矩会发生明显变化,影响发射波长的位移。通过收集不同波段的光,可以精确的定位不同极性的细胞器。(2)荧光分子与特异性靶向蛋白共价结合实现细胞器的定位成像实现活细胞内对特定蛋白质的实时动态分析是科学家一直探索的问题之一。SNAP-tag无论在体内还是体外都可以通过共价键与苯甲基鸟嘌呤衍生物发生快速特异性反应。当我们利用基因工程在某一细胞器上融合表达SNAP-tag蛋白,通过荧光探针与SNAP-tag蛋白的特异性结合就可以间接将荧光分子紧紧的带入特定的细胞器上,实现细胞器的定位与可视化。(2)基于酶切反应调控荧光信号实现特异性识别肿瘤蛋白酶作为许多疾病的重要诊断生物标志物,在调节各种代谢过程中起重要作用。体内监测蛋白酶对于生物学、医学及相关领域的研究至关重要,特别是在肿瘤中。γ-谷氨酸转移酶(GGT)是在实体瘤中过度表达的最重要的蛋白酶之一。所以准确识别GGT可以精准定位肿瘤。探针由荧光分子通过酰胺键与特定的识别基团相连,不发荧光。但当过量GGT存在的条件下,识别基团可以特异性与GGT反应,切断酰胺键,水解去除底物肽链,释放出荧光分子,发出荧光,实现实时、精准的定位肿瘤组织。更多还原

【Abstract】 Mechanisms of physiological and pathological changes are very important for the prevention and diagnosis of diseases.Cells,as the basic structural and functional units of living organisms,participate in various physiological processes.Accurately monitoring the formation process and microenvironment changes in cells is the basis for understanding the physiological and pathological changes.Due to the relatively low concentration of intracellular analytes and complicated envirounment in vivo,imaging tools with high sensitivity are urgently needed.Traditional imaging methods are lack of specificity and sensitivity,such as computed tomography,magnetic resonance imaging and ultrasound.Positron emission tomography is highly sensitive,however,limited by the strict safety regulation for radioactive compounds.Optical imaging such as fluorescence and bioluminescence imaging has resulted in accurate diagnosis through non-invasive,real-time and high-resolution imaging.Therefore,localization by fluorescence imaging has realized the visualization of specific organelles or whole living cell,providing a powerful tool for the accurate prevention and diagnosis of diseases.Following is the main content of this thesis:(1)Using the unique physical properties of specific organelles to locate and monitor the formation process of lipid dropletsAs an important physical parameter,polarity plays an important role in the change of cell physiological process.Compared to proteins,nucleic acids and carbohydrates,the lipid structures usually have extremely low polarity.However,fluorescent dyes(polarization-sensitive fluorescent probes)whose emission spectra change significantly with polarity can locate lipid substances.After electron excitation,the dipole moment of polarization-sensitive fluorescent probe changes obviously,which affects the shift of emission wavelength.By collecting emitted fluorescence at different wavelengths,organelles of different polarity can be clearly located.(2)Covalently bind specific targeted proteins with fluorescent chromophors for localization organellesReal-time dynamic analysis of specific proteins in living cells has attracted extensive attentions,recently.SNAP-tag can rapidly and specifically reacted with benzylguanine derivatives through covalent bonds both in vivo and in vitro.We used genetic engineering to fuse the SNAP-tag protein into different organelles.The designed fluorescent molecules with BG groups were brought into the specific organelles through specific binding of the BG group in fluorescent probe and SNAP-tag protein to achieve the localization and visualization of desired organelles.(3)localize tumors through monitoring specific enzymes by fluorescent probes with desired targeting groupsProtease,as an important diagnostic biomarker for many diseases,plays an important role in regulating various metabolic processes.In vivo protease monitoring is important for biological,medical and related research,especially in tumors.γ-glutamyl transpeptidase(GGT)is one of the most important proteases overexpressed in tumors.Therefore,accurate recognition of GGT can be used to locate tumors.Fluorescent probe with specific recognition moiety through the amide bond is colorless with barely fluorescent emission.After exissive GGT in tumors breaks the amide bond and releases the fluorescence molecule,strong fluorescence are emitted.With the designed probe,we can accurately target tumors in vivo.更多还原