【作者】 李军；

【导师】 吴水珠；

【作者基本信息】 华南理工大学 ， 材料学， 2016， 硕士

【摘要】 一氧化氮(NO)是非常重要的生物活性分子,其在生理学和病理学过程中起着至关重要的作用,NO由一氧化氮合酶(NOS)氧化L-精氨酸而生成。NO释放量紊乱会引发多种疾病,如在心血管系统中会引发动脉粥样硬化,高血压等；在神经系统中,其对脑部的成熟和发育,学习以及记忆等方面具有重要作用,当NO释放过量时,会引起老年痴呆病和帕金森综合症；在免疫系统中,NO却可以充当杀毒、杀菌作用,其不仅可以防御入侵人体的微生物和细菌等,而且还可以抑制癌细胞扩散。同样NO水平在肝中起着非常重要的作用,肝组织中NO水平异常会导致肝组织功能发生紊乱,如肝纤维化、肝硬化和肝癌等疾病。为了更好地理解NO的生理和病理功能,迫切需要具有灵敏度、能够在复杂生物系统中(如活细胞,肝等深层组织)定量检测NO水平的技术。与传统的检测方法相比,荧光检测技术因其灵敏度高、选择性好和低检测下限等优点被广泛应用在化学、材料、生物和医学等领域。目前主要有靶向检测线粒体和溶酶体中NO的荧光探针。尚未见报道靶向肝中NO的文献,然而NO与肝组织的正常工作有着密切关联,而且目前还未揭示生物学上NO在肝组织中的作用机制,主要由于缺少定位检测肝中NO的探针。鉴于上述情况,我们设计并合成了靶向定位检测肝中NO的增强型荧光探针Gal-RhB,并通过实验测试初步评估了其应用价值。该荧光探针是以罗丹明B为荧光基团,邻苯二胺为识别基团,接上半乳糖作为靶向定位肝的基团。同时接上半乳糖使该探针具有良好的水溶性,可以减小荧光探针与肝中的蛋白质等分子的非特异性结合。该荧光探针可以与肝表面的脱唾液酸糖蛋白受体(ASGP)特异性结合,使荧光探针可以检测生物体内肝中NO。在NO作用下,荧光探针Gal-RhB的邻苯二胺结构从罗丹明上断裂,罗丹明结构可以发生开环,荧光探针Gal-RhB检测液转变为粉红色。荧光探针Gal-RhB对NO具有高选择性和抗干扰性,具有良好水溶性,探针对pH的稳定性非常好,响应速度非常快,7分钟反应完全。从pH稳定性实验表明,该荧光探针可以适用于生理条件pH对NO的检测,当NO浓度在0-500 nM之间时,荧光探针在580 nm处荧光强度与NO浓度呈现出非常良好的线性关系,且探针Gal-RhB检测最低下限值为1.62 nM,检测灵敏度非常高。MTT实验表明探针Gal-RhB的细胞毒性小,可用于活细胞中对NO进行荧光成像。用细胞荧光成像结果表明：对肝癌细胞具有特异性靶向作用,探针能够检测肝癌细胞中NO。活体斑马鱼荧光成像表明：荧光探针Gal-RhB可以靶向定位斑马鱼中肝组织中NO。对于研究肝组织中NO作用机理具有非常重要的指导意义。更多还原

【Abstract】 Nitric oxide (NO) is an important biological messenger molecule, it plays an important role in the physiology and pathology, at the same time NO is generated from the oxidation of L-arginine under nitric oxide synthase (NOS). The disorder of NO can cause a variety of diseases. For example it will cause atherosclerosis and hypertension in the cardiovascular systems. In the nervous systems, NO is very important to brain maturation and development, learning, memory and other aspects, when the release of NO is excessive, it can cause alzheimer and parkinson. In the immune systems, NO can act as the role of antivirus and sterilization. Not only does NO can protect against the invasion of the microbe or bacteria, but also hinder the spread of cancer cells. At the same time the release abnormal of NO in the liver can cause various of hepatic diseases, such as liver fibrosis, cirrhosis, liver cancer and other diseases. To better understand the functions of NO in physiological and pathological, It is urgent to monitor the lever of NO with sensitivity and quantitative technology in biological systems (such as living cells, liver and other deep tissue). So monitoring the NO of the liver is very important to physiology and pathology. Compared to traditional detection methods, fluorescence sensor is widely used in the fields of chemistry, materials, biology and medicine because of its high sensitivity, good selectivity and low detection limit. There are some mitochondria-targeting and lysosome-targeting fluorescent probes for detecting NO, but no liver-targeting fluorescent probes for monitoring NO have been reported. However, the normal content of NO in liver closely linked to hepatic tissue. The mechanism of action of NO in liver has remained unraveled, mainly because of the lack of liver-targeting fluorescent probe for monitoring NO.In view of the foregoing, we have designed a liver-targeting fluorescent probe for monitoring NO, the reporter of the fluorescent probe is rhodamine B, at the same time o-phenylenediamine act as the recognition moiety, and galactose serves as the liver-targeting group. The fluorescent probe has a very good water solubility, the probe is selective towards NO, hence fluorescent probe can be used to detect NO. In the present of NO, o-phenylenediamine moiety of fluorescent probe Gal-RhB cleaved from the probe. So rhodamine will transform into the open-ring form, thus strong emission occurs.The water-soluble fluorescent probe of Gal-RhB can monitor NO with high selectivity and stability under different pH values. At the same time the response is very fast, and the reaction is completed in 7 minutes. When the concentration of NO ranges from 0 to 500 nM, good linear response can be obtained and the detection limit is 1.62 nM.MTT cytotoxicity experiments show that the probe of Gal-RhB is low cytotoxicity which can be used for imaging in living cells. Fluorescence imaging results showed that the probe is able to target hepatic cells and detect NO in the liver. Zebrafish imaging shows that the fluorescent probe can target hepatic tissue of the zebrafish for monitoring NO. It also has a deeply significance for further studying of the mechanism of NO in the hepatic tissue.更多还原