【作者】 武为；

【导师】 杨军；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 环境中的多种理化因素,包括紫外线、离子辐射和香烟烟雾等,都会对机体产生多种形式的损伤。特别是对于遗传信息载体-DNA的损伤,会引起DNA结构的改变和遗传物质的损失,即产生所谓的遗传毒性。近年来,随着这一领域的研究逐渐深入,人们对细胞内DNA损伤产生及响应机制已经有了一个初步的了解。但由于我们所知有限,这一领域还存在着大量悬而未决的科学问题,通过对这些问题的解答,将使我们在DNA损伤方面的认知得到进一步加深。热休克是否引起DNA损伤一直存在争议,我们实验室前期发现热休克不能诱导DNA双链断裂的特异性指标-γH2AX焦点的产生,但其它实验室却得到相反的结果。我们针对这一问题开展了进一步的研究,结果发现不同的热休克处理方式对产生的细胞毒性包括遗传毒性也各不相同,而不同的细胞系对热休克处理的响应也各不相同。45℃热休克引起细胞不同程度的死亡。在遗传毒性方面,五种热休克处理方式诱导细胞内γH2AX焦点产生的能力也各不相同,只有金属浴不论在42℃还是45℃下,FL细胞或是CHL细胞中都能够诱导γH2AX焦点的产生。两种细胞的表现方式也有所不同,CHL细胞对热休克处理的响应比FL细胞更加灵敏。而且我们发现γH2AX焦点产生与细胞存活率并无必然联系,因此γH2AX焦点不能作为热休克引起细胞死亡的判断标准。顺铂是一种临床常用的抗癌药物,主要用于生殖系统癌症和头颈部癌症的治疗,但其作用机制仍未明确。系统生物学的方法能够为我们提供一个高通量的、系统全面的研究平台。近年来,已经有多个研究小组利用蛋白质组学的方法对顺铂的细胞损伤机制进行了研究,但作为一种以DNA损伤为主的药物,引起的主要应答反应集中在细胞核内,因此我们利用蛋白质组学的方法对核蛋白组进行了表达谱分析,并最终筛选到了19个顺铂处理后引起表达量发生改变的蛋白质。这些蛋白包括核纤层蛋白、mRNA表达调控蛋白、细胞周期相关蛋白和生物大分子合成相关蛋白等,功能涉及细胞基本代谢和生理过程的多个方面。我们对其中部分蛋白进行了表达验证,并通过对Fas基因两种变异剪切产物的表达量检测,证实顺铂处理确实引起了HeLa细胞中变异剪切现象的发生。纳米技术是一种开发应用纳米材料的新型技术,纳米材料因其物理结构上的特殊性,在应用方面有着极大的优势,但它是否会对机体产生毒害作用尚未明确。量子点作为一种荧光纳米材料,广泛应用于细胞成像等生物医学领域。细胞存活率检测结果显示PEG包被的CdSe/ZnS核/壳量子点对细胞基本不产生毒害作用。我们同时对量子点的细胞毒性进行了蛋白质组学分析,共找到了21个差异表达的蛋白质点,占蛋白总数的0.5%。其中5个蛋白质点得到质谱鉴定,包括两个锌指蛋白、两个角蛋白和一个过氧化物酶。鉴于量子点处理后的HSF细胞中只有0.5%的蛋白质表达量受到显著影响,结合细胞存活率的检测结果,可以认为这种量子点的细胞毒性较小,是一种比较安全的纳米材料。更多还原

【Abstract】 Life could be threatened by a lot physical or chemical stresses in the environment, including ultraviolet,ionizing radiation and cigarette smokes.Such stress can cause damage,especially DNA damage in cells,which is called genotoxicity.Due to the extensive and insightful research about DNA damage in recent years,we have now acquired a rather large body of knowledge about the mechanisms involved in DNA damage induction and signaling.However,many questions remain unanswered,which warrant further detailed investigation.There have been controversies about whether heat shock could induce DNA damage.While we previously found heat shock could not induceγH2AX foci formation,which is regarded as a marker of DNA double strand breaks,Takahashi et al suggested otherwise.In order to clarify this discrepancy,the effects of five different heating methods onγH2AX foci formation were examined.We found that the cytotoxicity effects of different heating methods varied differently,and different cell types showed different heat shock responses.It was found that during a 24 h period, heating at 42℃for 30 min did not cause significant loss of cell viability,while 45℃caused various degrees of decrease in cell viability.The ability of these five methods to induceγH2AX foci was quite distinct,with only heat block inducedγH2AX foci at both 42℃and 45℃in both FL and CHL cells.The two cell types also respond differently to the different heating methods,with CHL being more sensitive to heat shock.However,expression ofγH2AX may not be used as a surrogate of cell killing.Cisplatin is an effective anti-cancer drug widely used in clinical treatment of reproductive system cancers as well as head and neck cancers;however,the underlying mechanisms are not fully clear yet.Systems biology approaches provide us new platforms to conduct study at whole genome or proteome level.Several groups have used proteomic technologies to explore the anti-cancer mechanisms of cisplatin,and identified some responsive proteins.However,as DNA is the primary target of cisplatin,and many cellular events induced by cisplatin took place in nucleus,we analyzed the nuclear proteome expression pattern after cisplatin treatment.A total of 19 proteins were eventually successfully identified among those differentially expressed proteins.These include nuclear lamina proteins,mRNA processing-related proteins,cell cycle-related proteins and synthesis-related enzymes,all of which were involved in basal metabolism and biological process in cells.The changed expression pattern of these proteins were further validated by Western blot or immunofluorescent microscopy,and cisplatin-induced alternative splicing was confirmed by RT-PCR analysis of Fas gene transcripts.Nanotechnology is a fast growing area,and nanomaterials have great application potentials due to their unique physical and chemical properties.However,the potential toxic effects of the nanomaterials are not clear,and it has attracted a lot attention recently.Quantum dots(QD) are semi-conductive nanomaterials that can be used for cell imaging.In this study,we also evaluated the potential toxic effects of quantum dots using proteomic approach.Results of cell survival test showed that poly(ethyleneglycol)-coated CdSe/ZnS core/shell quantum dots did not significantly affect the cell viability of human skin fibrobalsts(HSF).By proteomic analysis,a total of 21 proteins were found differentially expressed after 8 nM and 80 nM PEG-QD440 and PEG-QD680 treatment.MS analysis identified 5 proteins,including two zinc finger proteins,two keratins and Peroxiredoxin-2.Since 21 proteins only count for about 0.5%of the total proteins in HSF cells,it indicated that PEG-QD may not elicit significant adverse effects in cells.PEG-Qdots could be a promising agent in biological and medical applications.更多还原