生物荧光成像与人嗜中性粒细胞信号转导动力学的研究

【作者】 潘雷霆；

【导师】 许京军；

【作者基本信息】 南开大学 ， 光学， 2009， 博士

【摘要】 耗散结构理论在物理、化学、生物乃至社会等领域得到了广泛的应用,尤其是近年来在生命科学研究中的应用更是得到了重视,如在信号转导网络调控、免疫系统防御与杀伤调控、胚胎发育调控等研究。在本论文中,我们以耗散结构理论为指导,开展了人嗜中性粒细胞内烟酰胺腺嘌呤二核苷酸（磷酸）[Hydrogenated Nicotinamide Adenine Dinucleotide（Phosphate）,NAD（P）H]和钙(Ca²⁺)信号转导动力学的研究。人嗜中性粒细胞是免疫系统重要组成之一,其信号转导动力学过程具有耗散现象,如已发现NAD（P）H和Ca²⁺信号在细胞内转导过程呈现时空有序性的耗散模式。目前,活体细胞内信号转导过程中时空信息的有效获取主要依赖于活体荧光成像技术。因此我们构建了显微弱光高时空分辨率成像系统,可实现毫秒级信号连续采集,同时保证空间信息的获取。依托该系统,本论文在单个嗜中性粒细胞水平上研究了NAD（P）H自体荧光的探测问题,最终克服了NAD（P）H自体荧光强度较弱、易漂白淬灭,以及紫外激发光对细胞光毒性影响等难题,实现了该荧光信号在单个嗜中性粒细胞水平上的实时、快速、可视化成像,从而为研究单个嗜中性粒细胞内NAD（P）H信号转导动力学过程中的时空信息提供高时空分辨实验平台和有效技术支持。在此基础上,我们系统地研究了外源性一氧化氮（Nitric Oxide,NO）对佛波酯（Phorbol 12-Myristate 13-Acetate,PMA）激活的人嗜中性粒细胞内Ca²⁺动力学过程的影响。作为一种重要免疫信使分子,NO对嗜中性粒细胞趋化、粘附、呼吸爆发等生理功能的行使发挥着重要作用。同时,PMA是蛋白激酶C（ProteinKinase C,PKC）的特异性激动剂,可通过激活NAD（P）H氧化酶诱导嗜中性粒细胞呼吸爆发。我们的研究结果首次证明了外源性NO是通过亚硝基化反应途径诱导人嗜中性粒细胞内IP₃受体敏感的钙库释放,而不是通过传统的NO-sGC-cGMP信号途径,钙库释放可诱导明显的胞外钙内流,且整个过程是依赖于PMA诱导人嗜中性粒细胞的呼吸爆发。该发现为临床上使用外源性NO治疗相关免疫类疾病提供了崭新的思考视角及实验支持,也为研究细胞内Ca²⁺信号耗散现象提供了实验模型。此外,我们还应用PMA对类风湿关节炎模型的大鼠滑膜细胞进行了一定研究。类风湿关节炎发病机制与嗜中性粒细胞异常增殖密切相关。我们利用原子力显微成像术,使用PMA刺激滑膜细胞模拟炎症过程,结果显示PMA可以诱导大鼠关节炎模型的大鼠滑膜细胞骨架的明显隆起,并进一步揭示PKC激活可能是引起细胞骨架改变的重要原因,为加深了解类风湿关节炎的发生机制提供了实验基础。综上所述,依托构建的显微荧光成像技术,我们对嗜中性粒细胞内NAD（P）H和Ca²⁺信号转导动力学过程进行了研究,相关结果为进一步了解免疫作用机理和深入认识人体免疫防御系统的耗散现象提供了新的研究视角。更多还原

【Abstract】 Dissipative structure theory has extensively been applied in the research of physics, chemistry, biology and society. Especially, it is being studied in the field of life science including network regulatory of signal transduction, control of immune defence, control of embryonic development, and so on. In this paper, we studied the dynamics of hydrogenated nicotinamide adenine dinucleotide （phosphate） [NAD（P）H] and calcium (Ca²⁺) signal transductions in neutrophils with dissipative structure theory.Neutrophils are one of the body’s main components of immune system. Dynamics of NAD（P）H and Ca²⁺ signal transductions has been found to exhibit characteristic spatiotemporal patterns in such immunity processes of human neutrophils. Nowadays, the acquisition of spatiotemporal information depends mainly on the technique of living fluorescence imaging. Therefore, we constructed weak light micro-imaging system with high spatiotemporal resolution to study the autofluorescence NAD（P）H in single human neutrophils. The micro-imaging system can investigate fluorescence signals at about the milliseconds range with high spatial resolution. As a result, we achieved real-time, fast and visual imaging of autofluorescence NAD（P）H in single human neutrophils without running into the quenching or the phototoxicity problem due to the weak light micro-imaging system So that it is able to provide potent experimental support for studying spatiotemporal information in the dynamic process of intracellular NAD（P）H signal transduction.On this basis, we detailedly studied the role and signaling pathway of exogenous Nitric oxide （NO） in modulating Cytosolic calcium concentration ([Ca²⁺]_c) of phorbol 12-myristate 13-acetate （PMA）-activated neutrophils. NO, as an immunoregulatory messenger, has been reported that it can execute modulating in a wide variety of physiological functions and signal transductions in neutrophil chemotaxis, adhesion, phagocytosis, apoptosis, and oxidative burst. Meanwhile, PMA, a potent protein kinase C （PKC） activator, can induce respiratory burst of neutrophils by activating PKC, which subsequently activates NADPH Oxidase. To the best of our knowledge it is the first demonstration that exogenous NO induced calcium release from intracellular IP₃ receptor-sensitive stores and extracellular calcium influx via Snitrosylation reaction, but not via the activation of the NO-sGC-cGMP signaling pathway. More importantly, the effect of SNP on neutrophil [Ca²⁺]_c was dependent on the PMA-induced respiratory burst. Therefore, this study on modulation of neutrophils [Ca²⁺]_c by exogenous NO might be much more useful in the research of immunological disease. Meanwhile, it provides an ideal model to the research of dissipative patterns of [Ca²⁺]_c in neutrophils.In addition, this dissertation studied cytoskeleton change in rheumatoid arthritis （RA） rat Synoviocytes induced by PMA via the imaging of atomic force microscopy. The mechanism of RA correlates closely to the abnormal proliferation of neutrophils. PMA is usually used to simulate process of inflammation. The results showed that synoviocyte cytoskeleton changed significantly after PMA activation. The results also indicated that the activation of PKC might play crucial role in cytoskeleton changing of synoviocytes, which could help us to understand the mechanism of RA.In conclusion, we have made in-depth study of dynamic process of NAD（P）H and Ca²⁺ signal transductions in human neutrophils with our fluorescence micro-imaging system. The results are able to provide a new perspective for understanding the mechanism of immune response and dissipative patterns of body’s immune defense system.更多还原