【作者】 谭凤霞；

【导师】 王卫民； 王敏；

【作者基本信息】 华中农业大学 ， 水产养殖， 2008， 博士

【摘要】 水环境污染是全球关注的热点问题之一。全球每年都有大量的毒物通过各种途径进入水环境中,包括重金属在内的众多污染物对生物体都具有潜在的毒性。如果不能及时地发现毒物、检测其毒性以及对其潜在危害做出预测,那么极有可能造成严重的生态危机。因此建立灵敏、快速的生物检测体系对污染物的毒性进行早期预警和生态风险评估就显得极其重要。利用体外培养的细胞系作为体外模型进行污染物毒性检测的体外检测系统具有细胞均一性好、遗传相似、反应速度快、经济、方便等众多优点,使得其在毒理学中的应用越来越广泛。由于现在建立的鱼类细胞系的种类和数量繁多,而每种细胞系对毒物的反应又存在较大差异,因此,需要通过检测不同的污染物对不同细胞系的毒性,以确立敏感的、适合的细胞体外模型应用于其毒性早期预警的研究。本文主要研究鱼类细胞体外模型对检测我国水体中常见的四种重金属——镉（Cd）、铬（Cr）、锌（Zn）、铜（Cu）的毒性敏感性和适用性。通过比较不同培养代数、不同物种、不同组织来源的早期传代细胞系和永生性细胞系对重金属的毒性敏感性,确立每种重金属的相对敏感细胞系,然后以毒性敏感细胞系为体外模型检测重金属的细胞毒性、氧化损伤和基因毒性,探讨敏感的、适用的鱼类细胞体外模型在重金属毒性检测中的应用前景,主要研究结果如下:1、三株鱼类细胞系的建立利用组织块贴壁法新建了胭脂鱼（Myxocyprinus asiaticu）肌肉细胞系CSM与吻端组织细胞系CSSN以及稀有鮈鲫（Gobiocypris rarus）鳍条细胞系RMF三株细胞系,并通过细胞计数研究其最适生长条件和冻存复苏能力,通过染色体分析和线粒体基因分析鉴定其遗传种质。结果表明新建的三株细胞系均为成纤维贴壁型细胞,形态较均一;三株细胞系均能在含10%FBS的L-15培养液及25℃的培养条件下具有连续传代能力,其中CSM的最适培养液为M199;冻存的5^th、10^th、15^th细胞的复苏能力较强,其复苏后存活率均在90%以上,贴壁率在50%-80%之间;染色体分析表明新建细胞系虽然存在染色体缺失与增多现象,但仍为正常四倍体细胞（染色体条数100,CSM和CSSN细胞系）和二倍体细胞（染色体条数为50,RMF细胞系）;线粒体基因（12s rRNMl8s rRNA、cytb）分析表明PCR扩增的基因片段序列与其来源鱼类的发表序列完全一致。可见,新建的三株鱼类细胞系具有体外连续传代能力且未发生遗传突变,可作为早期培养细胞模型应用于水环境污染物的毒性检测。2、十二株鱼类细胞系对检测重金属毒性的敏感性研究（1）首先,利用MTT法（以IC50值为指标）和细胞总蛋白含量测定法（CB法）（以IC50值为指标）检测氯化镉（CdCl₂·2.5H₂O）、重铬酸钾（K₂Cr₂O₇）、氯化锌（ZnCl₂）、硫酸铜（CuSO₄·5H₂O）对草鱼CIK细胞的细胞毒性,利用彗星实验法（SCGE法）（以DNA损伤率为指标）以及PI染色流式细胞仪法（FCM法）（以细胞凋亡率为指标）检测镉、铬（Ⅵ）、锌、铜对草鱼CIK细胞的基因毒性。结果表明四种体外检测方法均能通过不同的指标定性比较重金属对草鱼CIK细胞的毒性大小,其检测出重金属的毒性大小均为Cd＞Cr＞Cu＞Zn;两种重金属细胞毒性检测方法——MTT法和CB法得到的四种重金属的IC50值无显著性差异;综合比较这四种方法的优缺点,确定了MTT法为简单、快速、重复性好的重金属毒性敏感细胞系体外检测方法。（2）其次,利用MTT法,以IC50值为指标,比较了三株不同物种、不同组织来源、不同培养代数的新建细胞系,以及来源于不同组织、不同物种的九株永生性鱼类细胞系对重金属毒性的敏感性。结果表明随着培养代数的增加,三株新建细胞系对重金属的毒性敏感性有所下降,但在20代之内未见显著性差异;同一物种不同组织来源的鱼类细胞系对重金属的毒性敏感性是不同的;同一组织来源的不同物种的鱼类细胞系对重金属的毒性敏感性也是不同的,且早期传代细胞系不一定比永生性细胞系对重金属的毒性更敏感;确定了氯化镉的毒性敏感细胞系为斑点叉尾鮰卵巢细胞系CCO,重铬酸钾和氯化锌的毒性敏感细胞系均为鲤上皮瘤细胞系EPC,硫酸铜的毒性敏感细胞系为草鱼肾脏组织细胞系CIK。3、毒性敏感细胞系在重金属毒性检测中的应用以上述研究确定的毒性敏感细胞系作为体外模型,通过细胞形态观察,MTT法和CB法检测重金属的细胞毒性,采用脂质过氧化水平、GPX和SOD酶活性的测定检测重金属造成的氧化损伤,利用彗星实验法检测DNA的损伤,使用流式细胞法检测细胞凋亡和细胞周期的变化。结果表明重金属能引起细胞发生变圆、崩解、脱落,并抑制细胞生长,显著降低细胞存活率,引起细胞脂质过氧化物水平增加,造成细胞GPX和SOD酶活性下降,总抗氧化能力减弱,导致细胞发生氧化损伤;使细胞DNA单链发生断裂,发生DNA损伤;并引起细胞凋亡和细胞周期时相改变,且这些作用均呈浓度依赖关系。可见,确定重金属毒性敏感细胞系进行其毒性早期预警是可行的,并且可以较为准确地预测重金属的毒性作用。更多还原

【Abstract】 Aquatic environmental pollution is one of the major issues of global concern. A large number of chemical pollutants enter the aquatic environment each year from various sources. These pollutants, which include some heavy metals, are known to be potentially cytotoxic to biota and present a health threat to the public. If the prevalence of toxicants is not discovered and their toxicity monitored timely to forecast the potential threat, these pollutants may be at the bottom of severe ecological crisis. Thus, establishment of a sensitive biological monitoring system for early detection and ecotoxicological evaluation is required. Applying cell cultures as in vitro biological model to ecotoxicological assessment offers a number of well-described advantages as compared to in vivo animal tests. Culturing established fish cells in vitro is relatively rapid, cost-effective, readily reproducible, and can be easily adapted to automated high-throughput screening technologies. Establishment of more and more cell cultures from marine and mammalian species has promoted the rapid development of cell cultures as sensitive acute bioassays for the assessment of toxicological risks associated with chemical pollutants worldwide. In the near future, it is anticipated that the utilization of fish cell lines as a biological model for evaluating the cytotoxicity of pollutant chemicals in environmental samples will become a standard practice. To facilitate the use of established in vitro fish cell lines as valuable biological tools to monitor and detect environmental pollutant chemicals, it is necessary to test more cell lines comparatively to identify the more sensitive cell cultures as bio-indicators to common toxic chemicals.In this paper, the sensitivity and applicability of twelve fish cell lines to detect the toxicity of the four common heavy metals cadmium （Cd）, chromium （Cr）, zinc （Zn） and copper （Cu）, which prevail in aquatic environment of China, were investigated. The toxicity and sensitivity of each heavy metal to the different passages, species and tissues of early passaging and permanent fish cell lines were compared to ascertain the relatively sensitive fish cell line against each heavy metal. Then the heavy metal induced cytotoxicity, oxidative damage and genotoxicity on the relatively sensitive fish cell line against each heavy metal were investigated to discuss the foreground of sensitive and applicable in vitro models to investigate the toxicity of heavy metals. The main results are as follows: 1 Establishment of three new fish cell linesThe Chinese sucker muscle cell line （CSM）, Chinese sucker snout cell line （CSSN） derived from Chinese sucker （Myxocyprinus asiaticu） and the rare minnow fin cell line （RMF） derived from rare minnow （Gobiocypris rarus） were established and their biological characterizations including the optimal growth conditions （temperature, the content of serum and media）, the ability of cryopreservation resuscitation, chromosome and genes from ribosome （12s rRNA/18s rRNA） and cytochrome b were identified. The results are as follows: three newly established cell lines were all fibrillose, homogeneous cells; the new cell lines had the ability of continuum passage in L-15 （CSM in M199 medium） with 10% FBS at 25℃; the new cell lines had the strong ability of cryopreservation resuscitation at the passage 5^th, 10^th, 15^th the viability and the adherent ratio after cryopreservation resuscitation were > 90% and 50% - 80%, respectively; chromosome analysis indicated these cell lines shared a major chromosomal peak of 100 （tetraploid number for CSM and CSSN） and 50 （diploid number for RMF）, only small proportion of heteroploidy and aneuploidy; mitochondrial gene analysis indicated the identified sequences from the cells DNA were identical to the respective sequences reported from their fish species. Therefore, the three newly established fish cell lines had the ability of continuum in vitro and no mutation, they could be to detect the toxicity of environmental pollutions as early cell cultures.2 Comparison of the toxical sensitivity to heavy metal among twelve fish cell lines（1） Firstly, the cytotoxicity of cadmium chloride （CdCl₂·2.5H₂O）, potassium dichromate （K₂C₂O₇）, zinc chloride （ZnCl₂） and copper sulfate （CuSO₄·5H₂O） to grass carp （Ctenopharyngodon idellus） kidney cell line （CIK） was as Cd > Cr > Cu > Zn determined by MTT assay （IC50） and CB assay （IC50）. Despite some minor variations, the IC50 - values obtained from MTT assay and CB assay appeared very similar, and no significant difference between these two methods. The genotoxicity of these four heavy metals were also in the aforementioned order determined by SCGE assay （the ratio of DNA damage） and FCM assay （the ratio of cell apoptosis）. Comparatively, the MTT assay was relatively rapid, cost-effective, readily reproducible, and could be easily applied to investigate the relatively sensitive fish cell line of each heavy metal.（2） Secondly, to establish the potential use of cell cultures as a simple and sensitive biological tool to detect toxicity of heavy metals, the different passages, species, and tissues of three newly established cell lines and nine permanent cell lines established from different fish species and tissues were tested and compared for their cytotoxic sensitivity by MTT assay. The results indicated that the cytotoxic sensitivity decreased along with the increase of cell passages, but no significant difference amongst 20 passages; the cytotoxic sensitivity were different among the different tissues from the same species; the cytotoxic sensitivity were also different among the different species from the same tissue origin, and the early passaging cell lines were not always more sensitive than the permanent cell lines invariably; CCO cells derived ovary of channel catfish （Ictalurus punctatus） were the most sensitive one to CdCl₂·2.5H₂O, EPC cells derived from epithelioma of common carp （Ctenopharyngodon cyprini） were more sensitive than other cells to K₂Cr₂O₇ and ZnCl₂, while CIK cells derived from kidney of grass carp were the most sensitive cell line to CuSO₄ˉ5H₂O.3 The application of toxically sensitive fish cell line in detecting heavy metal toxicityThe cellular damage including the configuration under inverted microscope, cytotoxicity by MTT and CB assays, oxidative damage by determination the content of MDA and the activities of GPX, SOD and T-AOC, the DNA damage by SCGE, the cell apoptosis and cell cycle by FCM assay of heavy metals on their each toxical sensitive cells were investigated. After the treatment of heavy metals, signicant increases of round, collapsed and desquamated cells, significant decreases in survival, significant decrease in activities of GPX, SOD, T-AOC, an increase in the content of MDA, increases the ratio of DNA damage and apoptosis and the change of cell cycle were observed in a dose-dependent manner. Shortly, it was feasible to confirm the relative sensitive fish cell line to each heavy metal for early detection and ecotoxicological evaluation because of the different sensitivity level of different fish cell lines to the same heavy metal.更多还原