【作者】 薛莲；

【导师】 周建华；

【作者基本信息】 苏州大学 ， 劳动卫生与环境卫生学， 2004， 硕士

【摘要】 目的：镉（Cadmium，Cd）是一种体内蓄积性很强的环境污染物，可以引起肾、肺、肝、睾丸、脑、骨骼及血液系统损伤，目前尚无治疗慢性镉中毒的有效药物。并且镉已被确定为人类致癌物。突变被认为在肿瘤发生的启动和促进阶段均具有决定性作用。本研究旨在全面了解镉体外致突变作用及其特点，探讨相关机制；进而研究镉的遗传毒性拮抗剂—锌的作用及机制，为今后镉环境暴露的预防工作提供一定的实验依据。 方法：本研究是在体外实验体系中，以中国仓鼠肺成纤维细胞（V79，为国际上通用的细胞株）为研究对象，在细胞毒性基础上采用克隆法研究不同剂量氯化镉（CdCl₂）对V79细胞hprt基因位点突变频率的影响；采用碱性单细胞凝胶电泳技术（SCGE），分别检测不同剂量CdCl₂对V79细胞DNA的损伤作用、对由过氧化氢（H₂O₂）所致DNA损伤后修复的影响以及CdCl₂所致的V79细胞凋亡的变化，凋亡同时还采用了AO／EB双标记法结合激光共聚焦显微镜技术进行分析。以上在检测CdCl₂单独染毒效应的同时还采用了生理浓度（10μmol／L）的氯化锌（ZnCl₂）与不同剂量CdCl₂同时作用于V79细胞，以观察ZnCl₂对于CdCl₂所致细胞毒性、hprt基因位点突变、DNA损伤、修复和凋亡作用的影响。最后，分别采用等离子体光谱仪技术以及活性氧（ROS）荧光探针结合激光共聚焦显微镜技术测定V79细胞在CdCl₂单独及CdCl₂与ZnCl₂同时染毒后细胞内Cd²⁺、Zn²⁺以及ROS的含量变化，以探讨镉的毒性机制以及锌的拮抗机制。 结论：CdCl₂可以引起V79细胞急、慢性细胞毒性；对V79细胞具有致突变作用，氯化福致突变作用和相关机制及拮抗剂的研究中文摘要锅所致的彻rt基因位点突变频率增高存在两个峰值，这是其他研究者未曾报道的。进一步研究发现，CdCI:的致突变作用可能是由于其对于V79细胞DNA的损伤、DNA修复的抑制和对细胞凋亡的作用所引起的，其中对于DNA损伤和修复的影响可能是造成突变的主要原因。生理剂量的ZnC12可以拮抗CdC12所致的细胞毒性和突变效应，并且可抑制由cdCI:所引起的DNA损伤、修复及凋亡作用，其中锌对福致突变作用的影响也未见报道。生理浓度的zncl:可能是通过减少cdz+进入细胞以及降低细胞内ROS的生成而发挥其拮抗作用的。因此，维持体内锌的正常生理浓度对于福的致突变毒性可能会有一定的预防作用。在本实验中还发现，SCGE法可灵敏、快速地进行环境毒物DNA损伤、修复和凋亡的检测;激光共聚焦显微镜则对于以图像为基础的定性、定量分析具有显著的优势。更多还原

【Abstract】 Objective: Cadmium is a ubiquitous environmental contaminant that has been evaluated as an assured human carcinogen. The main sources of cadmium exposure in human are tobacco smoke, food and industrial pollution. Cadmium has a long biological half-life of more than 25 years which makes it a cumulative toxin. Unfortunately, there’s no available treatment for chronic cadmium poisoning. Mutation has been regarding as crucial to the initiating and promoting stages of carcinogenesis. In the present study, we determine the mutagenicity of cadmium comprehensively, searching the corresponding mechanisms and the effects of zinc as an inhibitor in vitro. The aim of this study is to explore the basic mechanisms of carcinogenesis of cadmium and to provide experimental data for the future steps carried out to prevent the exposed population from the carcino-genicity of cadmium.Methods: An in vitro system with the Chinese hamster lung fibroblast cell,V79-an international accredited cell line, was taken in the present study, which contained four parts. The acute and chronic cytotoxicity of Cadmium chloride (CdCl2)on V79 cells, offering the bases for dosage in the latter parts; the change of hprt locus mutation frequency in V79 cells exposed to a wide range doses of CdCl2 DNA damage, effects on the process of DNA repair and apoptosis were further carried out in V79 cells treated with different concentrations of cadium by Alkli single cell gel electrophoresis (SCGE) , the apoptosis effects were also assessed by Ardine orange/ethidum bromide double fluorescent dyestaining combined with the Confocal Scanning Laser Microscope(CSLM). The inhibitive effects by physiological concentration of zinc chloride (ZnCl2) were investigated by combined treatment with 10mol/L ZnCl2 and various concentrations of cadmium simultaneously on V79 cells in the above three parts. In the last part, the mass spectrography and the fluorescent probe of ROS(DCFH-DA) combined with CSLM were used to find the intracellular Cd2+ and Zn2+ ions and ROS respectively after treatment of CdCl2 alone or with physiological concentration of ZnCl2 simultaneously to explore the direct and indirect mechanisms of cadmium toxicity and the ways of the inhibitive effects for inorganic zinc.Conclusion: CdCl2 can cause the acute and chronic cytotoxicity in V79 cells. The mutation frequency of hprt increases in V79 cells exposed to CdCl2 with two peak values could be detected, which hasn’t been reported by other researchers. The inducement of CdCl2 on hprt locus mutation can be attributed to the effects of CdCl2 on DNA break, the process of DNA repair and apoptosis, in which the facilitation of DNA break, inhibition of DNA repair process may be the main causes. Physilogical concentration of ZnCl2 can virtually inhibit all the above-mentioned effects of CdCb, the impact of ZnCl2 on hprt mutation caused by CdCl2 also hasn’t been published until now.The inhibitive effects of zinc may be due to the decrease of intracellular Cd2+ and ROS caused by zinc, so maintaining the normal level of humoral zinc may facilitate the protection from carcinogenesis of cadmium. It can also be concluded that SCGE is a rapid,economic and way to detect DNA damage, DNA repair process and apoptosis induced by the environmental toxins, while CSLM has the advantages in qualitative and quantitative analyses of the image-based biological and pathological effects.更多还原