【作者】 吴云霞；

【导师】 邢达； 陈群；

【作者基本信息】 华南师范大学 ， 光学， 2005， 硕士

【摘要】 细胞凋亡是机体生命活动中重要的细胞学事件,在许多疾病的治疗中也起着关键性作用。光动力学疗法(PDT)是一种治疗癌症的新的技术,由它所引起细胞的死亡多以凋亡的形式发生,但由PDT引起的细胞的凋亡机制及其内部的调节过程都很复杂。本论文在充分调研国际、国内有关PDT凋亡研究的历史和进展的前提下,将FRET技术和化学发光探针运用到PDT凋亡机理的研究,得到了初步但非常有意义的结果,证明这两种方法在凋亡机理研究中的可行性。 本文首先概述了有关光动力疗法的基本知识以及治疗肿瘤的机制,总结了细胞凋亡相关蛋白的基本信息以及与细胞凋亡通路的调节,并对FRET技术的基本原理以及在蛋白相互作用的应用情况做了简要的描述。 本论文分三个部分详细介绍了实验工作: 在第一部分的实验工作中,运用荧光共振能量转移(FRET)成像技术监测在肿瘤坏死因子(TNF-α)和光动力作用诱导凋亡过程中细胞内caspase-3的活性,并对在凋亡前后细胞内稳定表达的荧光蛋白发射光谱的改变及FRET探针稳定表达后在细胞内的分布情况进行了研究。实验结果显示:FRET探针稳定表达后在细胞中细胞核和细胞质中均有分布;且细胞在凋亡前后的荧光发射光谱有明显的变化,而PDT作用时的照射激光对探针的FRET效率并无影响。对细胞进行FRET成像分析发现:未经任何处理的荧光细胞中CFP和YFP的荧光强度无明显的改变:经TNF-α或PDT处理的细胞,荧光蛋白CFP/YFP的比率有明显的增加。荧光探针的CFP/YFP的上升反应了细胞内caspase 3活性的增强,说明不论是TNF-α还是PDT处理的细胞内,caspase-3的活性都有增强。因此,通过FRET探针成像的检测技术,可以在活细胞水平实时监测PDT过程中细胞内caspase 3活性改变以及凋亡的启动。 在第二部分的实验工作中,为研究Photofrin-PDT所引起的细胞凋亡的凋亡通路,对Photofrin的定位、caspase-8的活性及caspase-3活化的动力学过程进行检测。结果发现Photofrin结合到线粒体膜上,由于光敏作用导致的损伤主要发生在光敏剂结合位点附近几个纳米的范围内,因此线粒体为Photofrin-PDT诱导的凋亡的最初作用靶点。Photofrin-PDT处理后,caspase-8未被激活,它所诱导的更多还原

【Abstract】 Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. Photodynamic therapy (PDT) is a new cancer treatment modality and attracts many countries’ attention. In response to photodynamic therapy (PDT), apoptosis has been found to be a prominent form of cell death for many cells in culture. The mechanisms of the initiation and regulation of apoptosis induced by PDT are complex and diverse. So we investigated the status and progress of the PDT induced apoptosis, we used FRET technique and a specific chemiluminescence probe (FCLA) to study PDT-induced apoptosis. The results are primary but significative, suggesting that they are feasible methods for studying the mechanism of PDT-induced apoptosis.In this paper, the base information of PDT and the mechanism of ablating tumor are introduced firstly, and the characteristicsand the regulation of apoptosis are given in details. In addition, the principle and the application of FRET technique are described.According to the former research, we have done the following work.In the first experimental study, we studied TNF-a and PDT induced caspase-3 activation with fluorescence resonance energy transfer (FRET) technique. A recombinant caspase-3 substrate, SCAT3, was used as the FRET probe. FRET fluorescence images were collected after PDT or TNF-a induced apoptosis. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the caspase-3 activation started immediately after the PDT treatment. In contrast, FRET disruption caused by caspase-3 activation started at 3 hours after TNF-a treatment, due to different signaling pathway. The results have proofed, for the first time, that FRET is a sensitive technique that can be used to investigate PDT-induced activation of caspase-3 in real-time and in single cells. By choosing appropriate recombinant substrates as FRET probes, it is likely that FRET technique will provides a new real-time means to study the mechanism of PDT at single cell level.In the second experimental study, to determine the apoptosis pathway induced by Photofrin-PDT, we used the FRET probe SCAT3 to monitor the dynamics of caspase-3 activation after PDT treatment and the caspase-8 activity was measured. With laser-scanning confocal microscopy, we found that Photofrin can localize in mitochondria. The mitochondria are the primary targets of Photofrin-PDT. After PDT treatment, caspase-3 was activated rapidly while caspase-8 was not activated. The results indicated that PDT-induced apoptosis was initiated from mitochondria pathway and independent on caspase-8 activation. As a parallel study to confirm and compare apoptosis initiated by the death receptor pathway, the activation of caspase-3 was induced by TNF-a. TNF-α acts by binding to its receptors and recruiting components of death-inducing complexes that directly activate caspase-8. Compared with PDT-induced apoptosis, the onset of caspase-3 activation was delayed 3 hours and the caspase-3 activation was required a significantly longer time. The differential kinetics of the FRET probe cleavage in cells with TNF-α or PDT suggests that the different dynamics of caspase-3 activation are induced by these two-apoptotic stimuli. The results have proofed that the initiation and process of caspase-3 activation are different in response to different apoptosis pathways, PDT-induced apoptosis is directly initiated from mitochondria pathway and not involved in the death receptors-dependent pathway and the apoptosis progress initiation from the death receptors pathway is slower than that from the mitochondria pathway.In the third experiment study, we reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. In the experiment, we found that both HpD and FCLA could permeate through cellular membrane and localize to cytoplasm. Although the molecular weight of HpD is close to FCLA’s, the permeating efficiency of HpD through membrane is obviously differe更多还原