热休克、顺铂和量子点的遗传毒性以及对蛋白表达的影响

发布时间：2018-10-05 11:00

【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。
[Abstract]:Many physical and chemical factors in the environment, including ultraviolet radiation, ion radiation and cigarette smoke, can damage the organism in many forms. In particular, damage to the DNA vector-DNA can cause a change in DNA structure and loss of genetic material, that is, to produce so-called genotoxicity. In recent years, as the research in this field is in-depth, there has been a preliminary understanding of the mechanism of DNA damage generation and response in cells. But because of our limited knowledge, there is still a lot of outstanding scientific problems in this area, and through answering these questions, we will further deepen our knowledge of DNA damage. Whether heat shock causes DNA damage has always been controversial. We found that heat shock could not induce DNA double-strand break specific index in the early stage of our laboratory. As a result of this study, we conducted a further study of this problem, and the results showed that different heat shock treatments had different genotoxicity, and the response of different cell lines to heat shock treatment was different. In terms of genetic toxicity, the ability of five types of heat shock treatment to induce the focus of H2AX in cells is different, and only the metal bath can induce H2AX coke in FL cells or CHL cells, whether at 42 & deg; C or 45 & deg; C The effect of CHL cells on heat shock treatment was higher than that of FL cells. It was even more sensitive. And we found that the focus of H2AX was not linked to the survival of cells, so that the focus of H2AX was unable to cause cell death as heat shock. Judgment standard. Cisplatin is a commonly used anti-cancer drug, mainly used in the treatment of reproductive system cancer and head and neck cancer, but Its mechanism of action remains unclear. The system biology approach can provide us with a high throughput, In recent years, a number of research groups have used proteomics methods to study the mechanism of cell damage in cisplatin, but as a drug based on DNA damage, the main response is The reaction was concentrated in the nucleus, so we used the method of proteomic analysis to analyze the nuclear protein group, and finally screened the expression of 19 cisplatin. The protein comprises nuclear fiber layer protein, mRNA expression regulatory protein, cell cycle related protein and biological macromolecule synthesis related protein and the like, and the function relates to cell basic metabolism and In many aspects of the physiological process, we have demonstrated the expression of some of these proteins, and confirmed that cisplatin treatment did give rise to HeLa cells by detecting the expression of two variants of Fas gene. The occurrence of variation shear phenomenon. Nanotechnology is a new type of technology for the development of nano-materials. Nano-materials have great advantages in application because of its particularity in their physical structure, but whether it and the quantum dot is used as a fluorescent nanometer material, and is widely used as a fluorescent nanometer material. Applied in the biomedical field of cell imaging and the like, and the cell survival rate detection results show that the PEG-coated CdSe/ ZnS core/ shell quantity At the same time, the cytotoxicity of quantum dots was analyzed by proteomic analysis, and 21 differentially expressed eggs were found. The white particles accounted for 0.5% of the total protein. Among them, 5 protein spots were identified by mass spectrometry, including two proteins. Two keratin and one peroxidase. In view of the significant effect of only 0.5% of the protein expression in the HSF cells treated with quantum dots, the cytotoxicity of the quantum dots can be considered to be less toxic than the detection results of cell survival.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

【共引文献】