识别自由基的新型荧光探针研制及其用于单细胞成像和流动注射分析

【作者】 胡继西；

【导师】 唐波；

【作者基本信息】 山东师范大学 ， 分析化学， 2005， 硕士

【摘要】 在生物代谢过程中不断产生各种自由基,如:超氧阴离子自由基(O2-.)、羟自由基(HO·)、双氧水(H2O2)、一氧化氮(NO)和脂自由基(ROO·)等。其中羟自由基(HO·)是生命活动过程中产生的一种目前已知氧化性最强的自由基,它能造成组织过氧化、蛋白质交联变性、核酸损伤和多糖分解等,是目前公认的毒性最大的自由基。并且它产生的二级自由基会引起细胞,组织等不可逆转的损伤。因此在生命科学、环境科学等领域,已对HO·展开了大量研究。NO 是目前在体内发现的最小、最轻、最简单的生物信息分子,过去曾被称为内皮细胞衍生舒张因子(EDRF,endothelium-derived relaxing factor),在19 世纪80 年代,NO 被证明和EDRF 在产生的量和时间及生理功能具有同一性。体内NO 是NO 合酶(NOS)催化氧化L-精氨酸(L-Arg)生成的,它的生理作用与心血管系统、神经系统、泌尿系统、免疫系统和性兴奋都有密切的关系, Furchgott, Ignarro 和Murad 因研究发现NO 是心血管系统中的信息分子而共同获得1998年诺贝尔医学和生理学奖,此后各国都对NO进行了广泛的生物学研究。然而,由于生物体内活性自由基的寿命非常短,导致通常情况下稳态浓度极低,而短寿命和低浓度意味着自由基的测定异常困难,特别是在生物活体内(in vivo)与直接原位(in situ)的检测目前尚未有突破。另外生物体内还存在着预防自由基损伤的内在抗氧化防护体系,因而至今为止人们尚无法直接动态观测到某些器官中活性自由基的产生,对它们的生成机制,生理作用及其动态损伤生物机体的过程都缺乏足够的认识和充分的实验证据,以至在临床上直接影响和限制了对许多疾病的治疗效果。因此,针对动、植物生物活体内自由基开创新的分析与检测技术是分析化学学科发展中具有挑战性的前沿课题之一。荧光法具有简单、易行、灵敏度高与选择性好等特点。流动注射分析法重现性好,分析速度快,可以实现样品的实时、在线分析及分析过程的自动化。将流动注射与高灵敏度的荧光法结合起来用于测定自由基,有望实现生物样品中自由基的实时、在线、高通量分析,方法不仅具有很高的灵敏度和选择性,而且能够实现分析过程的自动化。本论文基于一氧化氮自由基和羟自由基与荧光探针作用后荧光光谱性质的改变,开展了以下几方面的工作。更多还原

【Abstract】 Numerous free radicals are generated in the course of biology metabolism, such as: superoxide anion radicals (O2-·), hydroxyl radicals (HO?), hydrogen peroxide (H2O2) and ROO·. The hydroxyl radicals, the strongest oxidants to be well known, are thought be generated within cells and tissues, where it can attack proteins, lipids and DNA. More important, it can initiate secondary radicals that can produce irreparable damages. Therefore, hydroxyl radicals have been suggested to play a critical role in many pathological processes. Nitric oxide was found to be smallest, lightest, simplest biological information molecule in the body, which was named endothelium-derived relaxing factor (EDRF) in the past. In 1980’s, it was proved that nitric oxide function as endothelium-derived relaxing factor in the body. Nitric oxide was synthesized through L-arginine catalyzed by nitric oxide synthase (NOS) in the cell, whose physiology action has an intimate relation with cardiovascular system, nervous system, urination system, immune system and venereal excitation. Furchgott, Ignarroand and Murad, who found that nitric oxide was an information molecule in the cardiovascular system, was award Nobel Prize in Physiology or Medicine in 1998.Therefore, biological research on nitric oxide was performed in many countries. It is well known that hydroxyl radical and nitric oxide have damages on organisms and have gotten great attention in the 20th century, but the short lives and low concentration of free radicals mean to the determination difficult, especially the determination in vivo and in situ has no breakthrough. In addition, there exist the anti-oxidation systems preventing the damages of free radicals in organisms. Up to date, it cannot be observed the dynamic generation of reactive oxygen species in some organs. Enough cognition and sufficient experimental evidences of their generation mechanism, physiological action and dynamic damages on organisms are not acquired. Therefore, the therapy effects of many diseases in clinic are limited and they are main obstacles to the plant cultivation in adversity. So it is one of challenging scientific researches in analytical chemistry, in which novel analysis and determination更多还原