【作者】 景毅；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2010， 硕士

【摘要】 海洋污染是全球关注的焦点问题之一。全球每年都有大量的有毒物质通过各种途径进入海洋中,包括内分泌干扰物在内的众多环境污染物对海洋生物体都具有潜在的毒性。内分泌干扰物(endocrine disruption chemicals, EDCs)是指具有干扰生物机体正常分泌物质的合成、释放、运转、代谢、结合等过程,激活或抑制内分泌系统功能,从而破坏维持机体稳定性和调控作用的物质。它包括天然存在和人工合成的一些化合物。内分泌干扰物在环境中虽然含量很少,但却具有很强的毒性效应,影响着人和动物的生殖、发育等过程。关于内分泌干扰物的研究已成为当今科学界的一个热点。体外培养的细胞系作为体外模型进行污染物毒性研究具有均一性好、遗传相似、反应速度快、经济、方便等众多优点,使得细胞系在毒理学中的应用越来越广泛。本文选择具有极高经济价值的海水养殖品种条斑星鲽(Verasper moseri Jordan et Gilbert),建立其卵巢组织细胞系,并以此细胞系作为体外研究模型,进行内分泌干扰物的生殖与遗传毒性研究。为建立条斑星鲽卵巢细胞系,本文根据条斑星鲽卵巢组织的结构特点,采用0.25%透明质酸酶和0.2%Ⅱ型胶原酶联合消化法启动了卵巢组织的原代培养。通过对比不同培养体系下的卵巢组织细胞的迁出及生长状况,确立了条斑星鲽卵巢组织细胞最适培养温度为22℃,最适培养基为添加有100μg/ml羧甲基壳寡糖、10ng/ml碱性成纤维样生长因子(bFGF)和40ng/ml I型胰岛素样生长因子(IGF-I)的20%胎牛血清(FBS)-DMEM/F-12培养液(pH7.2)。在此最适培养条件下,条斑星鲽卵巢组织原代启动5d后即有细胞迁出,为典型的成纤维样细胞形态,且生长分裂旺盛,15d即可长成汇合的细胞单层,并能稳定连续传代。细胞生长曲线分析结果显示,第60代的条斑星鲽卵巢细胞的群体倍增时间为59.7h。染色体数目与核型分析结果显示,第60代条斑星鲽卵巢细胞特征染色体数目仍为46条,且具有典型的条斑星鲽正常二倍体核型特征(44t+2sm),确为条斑星鲽细胞系。目前,该细胞系已连续继代培养至第65代,细胞生长分裂状况依然良好,已成功建立了条斑星鲽卵巢细胞的连续性细胞系(BFO)为了利用条斑星鲽卵巢细胞系细胞研究内分泌干扰物对卵巢细胞的毒性作用,本文选用镉(CdCl2)、铅(PbCl2)和多氯联苯(Aroclor1254)三种代表性内分泌干扰物,分别以不同的浓度对卵巢细胞进行了处理,利用光镜观察和MTT染色方法研究了三种内分泌干扰物对条斑星鲽卵巢细胞的毒性作用。结果显示,10μmol/L的CdCl2和20μmol/L的PbCl2对条斑星鲽卵巢细胞均能产生显著的抑制作用,且均呈浓度依赖性。低浓度(0.1-10μg/L)多氯联苯-1254对条斑星鲽卵巢细胞的增殖具有促进作用,但高浓度(10001μg/L)时反而会对卵巢细胞的增殖具有显著的抑制作用。表明三种内分泌干扰物对条斑星鲽卵巢细胞系细胞均具有显著的毒性作用,故对条斑星鲽具有显著的生殖毒性。为了确定三种内分泌干扰物对条斑星鲽卵巢细胞系细胞毒性作用的机理,本文又利用专用检测试剂盒对细胞生物氧化还原系统标志酶活性的影响作用进行了研究。检测结果显示,40μmol/L CdCl2和80μmol/L PbCl2处理24h即可引起条斑星鲽卵巢细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性的降低,以及丙二醛(MDA)含量的升高,表明40μmol/L CdCl2和80μmol/L PbCl2均能对条斑星鲽卵巢细胞产生显著的浓度依赖性的氧化损伤作用,而多氯联苯1254只有在1000μg/L的高浓度下才会引起卵巢细胞的氧化损伤。表明CdCl2、PbCl2和多氯联苯-1254三种内分泌干扰物对条斑星鲽卵巢细胞均具有显著的毒性作用。表明三种内分泌干扰物对条斑星鲽卵巢细胞毒性作用主要是通过引起卵巢细胞发生氧化损伤来实现的。为了进一步确定重金属对条斑星鲽卵巢细胞氧化损伤作用的最终结果,本文对CdCl2和PbCl2诱导条斑星鲽卵巢细胞系细胞凋亡的作用进行了研究。光镜观察结果显示,40μmol/L的CdCl2和μmol/L的PbCl2处理卵巢细胞24h之后即可诱导卵巢细胞凋亡,且细胞凋亡的程度有浓度依赖性；吖啶橙/溴化乙锭双染色结果显示,在40-160μmol/L的CdCl2和80-320μmol/L的PbCl2处理卵巢细胞24h之后均诱导卵巢细胞凋亡率分别为30%-80%和20%-70%；细胞DNA的琼脂糖凝胶电泳结果显示,80μmol/L和160μmol/L处理卵巢细胞24h之后可造成明显的DNA段片化。上述实验结果表明,重金属CdCl2和PbCl2诱导条斑星鲽卵巢细胞系细胞氧化损伤作用的最终结果是引起条斑星鲽卵巢细胞发生细胞凋亡。为了进一步研究内分泌干扰物CdCl2的遗传毒性,本文利用单细胞凝胶电泳法(single cell gel electrophoresis, SCGE)研究了不同浓度CdCl2对条斑星鲽卵巢细胞系细胞DNA的损伤作用。凝胶电泳检测结果显示,20μmol/L的CdCl2即可引起卵巢细胞DNA出现损伤,且其对DNA的损伤程度具有浓度依赖性,初步表明CdCl2对条斑星鲽卵巢细胞系细胞有较强的DNA损伤作用,故对条斑星鲽具有显著的遗传毒性。综上所述,本文成功建立了条斑星鲽卵巢组织细胞系,三种内分泌干扰物通过引起细胞发生氧化损伤对该细胞系细胞均具有显著的毒性作用,重金属能最终引起卵巢细胞的凋亡,证明三种内分泌干扰物对条斑星鲽具有显著的生殖毒性、CdCl2还具有较强的遗传毒性。本文所建立的条斑星鲽卵巢细胞系为研究内分泌干扰物对条斑星鲽等鱼类的生殖毒性和毒性作用建立了一个理想的体外实验体系,实验结果为揭示内分泌干扰物的毒性作用机理提供了理论依据,并为包括内分泌干扰物在内的环境污染物的毒性作用及致毒机理的研究奠定了基础。更多还原

【Abstract】 Ocean environmental pollution is one of the major issues of global concern. A large number of chemical pollutants enter the ocean each year from various sources. These pollutants, including endocrine disruptors, are known to be potentially cytotoxic to biota and present a health threat to the public. Endocrine disruption chemicals (EDCs) have been confirmed to be a group of particular environmental contaminants, including some natural and artificial compound. It’s extremely harmful to the reproduction and inheritance of organisms, though the content is very low in environment. The pollution of EDCs has raised increasing concern from every walk of life. It is required to establish a sensitive biological monitoring system for early detection and ecotoxicological evaluation. Cultured cells, as a sensitive bioassay, are relatively rapid, cost-effective and readily reproducible. It can be easily adapted to automated high-throughput screening technologies. Establishment of more and more cell lines from marine and mammalian species has promoted the rapid development of cell lines in assessing the toxicological risk associated with chemical pollutants worldwide.Although many fish cell lines have been established, few cell lines have been established from commercial marine fishes. This study was conducted to establish ovary cell line from barfin flounder, Verasper moseri, and to verify its application in the detection of EDCs toxicity.Ovary tissue from V. moseri was digested with 0.25% hyaluronidase and 0.2% collagenaseⅡto initiate the primary cell culture in Dulbecco’s Modified Eagle Medium:Ham’s Nutrient F-12(1:1) (DMEM/F12) medium supplemented with 20% fetal bovine serum(FBS), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and carboxymethyl-chitooligosaccharide at 22℃. The ovary cell in primary culture was in fibroblastic morphology and proliferated to confluence within 15 days. Growth property studies indicated that the ovary cell had population doubling time of 59.7 hours at passage 60. Karyotype analysis showed that the ovary cell exhibited chromosomal aneuploidy with a modal chromosome number of 46 and typical characteristics of the normal diploid karyotype (44t+2sm), which implied that the ovary cell was indeed from V. moseri. To date, barfin flounder ovary cell has been subcultured up to passage 65 and still grows in a good proliferating status. The continuous barfin flounder ovary cell line named BFO cell line has been successfully established.In order to research the cytotoxicity and oxidative damage to ovary cell from EDCs, cadmium chloride(CdCl2), lead chloride(PbCl2) and polychorinated biphenyls(Aroclor 1254), were chosen to treat BFO cells with different concentrations. The acute cytotoxic effects of EDCs were evaluated by Morphological observation and MTT assay. The results showed that 10μmol/L CdCl2 and PbCl2 were toxic to BFO cells in a concentration-dependent manner. While only high concentration of Aroclor 1254 (1000μg/L) was toxic to BFO cells. After treated by EDCs, BFO cells became round and lysed, and began to detach and finally died within several hours.After treated with the 40μmol/L CdCl2,80μmol/L PbCl2 and high level Aroclor 1254 (1000ug/L), the activities of superoxide dismutase(SOD), glutathione peroxidase (GSH-Px) and malondialdehyde(MDA) in BFO cells were influenced in a concentration-dependent manner. It suggested that endocrine disruptors cause oxidative damage of BFO cells.In order to detect the effects of EDCs (CdCl2 and PbCl2) on the apoptosis of BFO cells, further research was carried out by AO/EB double-fluorescent staining and DNA electrophoresis. After treated with 40-160μmol/L of CdCl2 and 80-320μmol/L of PbCl2 respectively for 24h, cultured BFO cells underwent apoptosis, the respective apoptosis rates are 30%-85% and 20%-70%. The apoptotic extent was in a concentration-dependent manner.In order to detect the genotoxicity of EDCs, the DNA damage caused by CdCl2 to BFO cells were determined by single cell gel electrophoresis (SCEG) assay. After treated with 20μmol/L of CdCl2 for 24h, cultured BFO cells showed DNA damage, and the extent was in a concentration-dependent manner. It suggested that CdCl2 have intensive genotoxicity to BFO cells.Through the detection of reproductive toxicity and genotoxicity of EDCs to the established ovary cell line from barfin flounder, we can conclude that the cell line can be used as a useful tool for the research of environmental toxicology.更多还原