基于蚯蚓体腔细胞的碳纳米管和五氯酚钠复合毒性效应研究

发布时间：2018-03-24 17:01

本文选题：碳纳米管　切入点：五氯酚钠　出处：《南京大学》2015年硕士论文

【摘要】：碳纳米管(Carbonnanotubes,CNTs)自1991年被发现以来,以其独特的理化性质和较好的生物相容性被广泛研究和利用。与此同时,CNTs也大量进入环境中,对生态系统造成的影响日益增加,其毒性研究也逐渐受到重视。五氯酚(pentachlorophenol,PCP)及其钠盐作为一种除莠剂和杀虫剂,在上世纪九十年代之前被大量使用,虽已停产并禁用,但由于其在环境中不易降解,目前还有大量PCP残留在环境中,有机污染物PCP与大分子材料CNTs复合后又会产生新的毒性效应。赤子爱胜蚓(Eisenia foetida)作为土壤生态系统中的标准受试生物,已有CNTs对其毒性的研究,但蚯蚓在个体水平上的毒理学试验耗时长,实验动物用量大,且灵敏度不高,其实际应用非常有限。而细胞试验耗材少、速度快、灵敏度高,且细胞的毒性指标与个体毒性指标又有一定的相关性,可在很大程度上反应个体水平的毒性。本研究对探索了赤子爱胜蚓体腔细胞的体外培养方法,并通过体外染毒,研究了 CNTs和PCP-Na单独或联合作用下对蚯蚓细胞的急性毒性、氧化损伤、基因损伤和膜结构损伤的影响。同时研究了 CNTs和PCP-Na对赤子爱胜蚓的生殖毒性试验,从多层面探讨两种受试物的毒性机理。本研究主要结果如下:(1)赤子爱胜蚓体腔细胞在体外可以悬浮状态进行原代培养。用96孔板培养时,最佳培养条件为温度25℃、培养基为RPMI-1640培养基、不通CO2、细胞浓度为2-4×104个/孔(1-2×105个/mL),培养时间为24h。细胞存活率可达100%。(2)赤子爱胜蚓体腔细胞体外培养染毒,PCP-Na的LC50为104.46mg/L,其毒性介于蚯蚓个体人工土壤试验染毒第7天和第14天急性毒性之间。而与浓度为50mg/L的CNTs复合染毒试验中,直接添加复合染毒LC50为109.39 mg/L,CNTs吸附PCP-Na后染毒LC50为112.92 mg/L,两种方法复合染毒后急性毒性都略有减小。碳纳米管急性毒性非常小,试验中未测得其LC50值,这与个体水平上的急性毒性试验结果一致。(3)碳纳米管与五氯酚钠都对细胞产生氧化损伤,产生ROS,并能引起脂质过氧化,生成MDA。低胁迫下,细胞能作出应激反应,SOD活性增大,高胁迫下,SOD活性降低。CAT是最不敏感的生物标志物。这与个体水平上研究结果类似。(4)用彗星试验评价蚯蚓受两种受试物的胁迫作用,较为灵敏。试验结果显示,各染毒组与对照组相比均有显著性差异,碳纳米管单独染毒时,其与DNA损伤有明显的剂量-效应关系。而五氯酚钠单独染毒时,浓度较高的染毒组DNA损伤反而较小。CNTs与PCP-Na两者复合染毒时,其遗传毒性大于受试物单一染毒时的毒性,表现出一定的协同作用。试验结果与个体水平上彗星试验的结果类似。(5)高浓度五氯酚钠、50mg/L碳纳米管组以及复合染毒组均能对细胞的膜结构造成损伤,增加细胞膜的通透性,并降低细胞的线粒体跨膜电位(Mitochondrial Membrane Potential,MMP)。五氯酚钠对膜的损伤比较大,两者复合染毒后,对细胞膜的毒性降低,两种污染物呈拮抗作用。受试物对细胞膜结构的损伤程度与MDA含量有一定的相差性,表明对膜结构的损伤主要是氧化损伤。(6)个体水平上生殖试验结果表明,游离态的碳纳米管可能会对蚯蚓生长起到一定的促进作用。高浓度的五氯酚钠具有较强的生殖毒性,会明显减少蚯蚓蚓茧的产量,低浓度的五氯酚钠对蚯蚓生长的影响较大,但做为补偿,蚯蚓的平均产茧量有所提高。碳纳米管对蚯蚓的生殖毒性较低,其与五氯酚钠复合染毒时,五氯酚钠的生殖毒性也降低,且吸附后添加的生殖毒性比直接添加的要低,表明是碳纳米管的吸附作用降低了游离态五氯酚钠的浓度,从而降低其生殖毒性。综上所述,细胞毒性试验各指标都要比个体水平指标灵敏,且与个体水平毒性测定结果有一定的正相关性。细胞毒性试验有望取代蚯蚓个体水平的毒性试验成为新的快速、高效、灵敏的研究污染物对土壤生物毒性的方法。
[Abstract]:Carbon nanotubes (Carbonnanotubes, CNTs) since 1991 were found, with its unique physicochemical properties and good biocompatibility has been widely studied and used. At the same time, a large number of CNTs into the environment, the impact on the ecological system is increasing, the toxicity of gradually attention. Pentachlorophenol (pentachlorophenol, PCP) and its sodium salt as herbicides and pesticides, is widely used in the last century until 90s, although discontinued and disabled, but because of its in the environment is not easy to degrade, there are still a large number of PCP residues in the environment, organic pollutants PCP and CNTs composite macromolecular materials will produce toxic effects of new pure. Earthworm (Eisenia foetida) as a soil ecosystem in the standard test organisms, study on the toxicity of the existing CNTs, but the toxicological test of earthworms in the individual level of time-consuming, experimental animal A large quantity, and the sensitivity is not high, its application is very limited. But the cell test material consumption, high speed, high sensitivity, toxicity index and toxicity index and individual cells have certain relevance, can reflect the individual level to a great extent. This study explores the toxicity of Eisenia foetida coelomic culture method cells in vitro, and by in vitro, study the acute toxicity on earthworm cells CNTs and PCP-Na alone or under the combined effect of oxidative damage, damage and genetic damage of membrane structure. At the same time on the CNTs and PCP-Na of the pure reproductive toxicity test of earthworm, investigate two chemicals toxicity mechanism from multi level. The main results of this study are as follows: (1) Eisenia foetida coelomic cells in vitro were cultured in suspension culture. With 96 hole plate, the optimum culture conditions for the temperature of 25 DEG C, RPMI-1640 medium Not radical, CO2, cell concentration of 2-4 * 104 / hole (1-2 x 105 /mL), the incubation time for 24h. cell survival rate of 100%. (2) Eisenia foetida coelomic cells cultured in vitro were PCP-Na LC50 104.46mg/L, the toxicity between individual earthworm artificial soil test after seventh days and fourteenth day acute toxicity. The concentration of CNTs and 50mg/L in the composite exposure test, directly adding composite LC50 exposure was 109.39 mg/L, CNTs after the adsorption of PCP-Na by LC50 was 112.92 mg/L, two kinds of methods of composite after exposure to acute toxicity are slightly reduced. The acute toxicity of carbon nanotubes is very small, the test was not detectable in the LC50 value, acute the toxicity test results and the level of the individual. (3) carbon nanotubes with sodium have oxidative damage to cells to produce ROS, and can cause lipid peroxidation, generate MDA. low stress, cells can make the stress response, increase the SOD activity, high Under the stress of SOD, decreased the activity of.CAT is the least sensitive biomarkers. The individual level and the results of similar studies. (4) evaluated by comet assay of earthworm by two kinds of test stress, is more sensitive. The test results show that the groups compared with the control group were significantly different, carbon nanotubes single exposure, the injury has the obvious dose effect relationship with DNA. And pentachlorophenol single exposure exposure group, the higher concentration of DNA damage instead of small.CNTs and PCP-Na combined exposure, the genetic toxicity was greater than the toxicity test of single exposure, synergistic effects. The results of test results and the individual level of the comet assay. (5) high concentration sodium pentachlorophenate, 50mg/L carbon nanotube composite group and exposure group can cause damage to the cell membrane structure, membrane permeability increase, and reduce cell mitochondrial The membrane potential (Mitochondrial Membrane Potential, MMP). Sodium pentachlorophenate on membrane damage is relatively large, the two composite after exposure, reduce the cell membrane toxicity of two kinds of pollutants showed antagonism. The tested damage of cell membrane structure has certain difference with the content of MDA, shows that the damage is mainly on the membrane structure oxidative damage. (6) the level of the individual reproductive test results showed that free carbon nanotubes may be to promote the growth of earthworm. High concentrations of PCP has strong reproductive toxicity, will significantly reduce the earthworms cocoon yield, effect of low concentration of pentachlorophenol on earthworm growth greatly, but as compensation, average cocoon production amount of earthworms increased. Carbon nanotubes reproductive toxicity on earthworm was low, the composite with sodium exposure, reproductive toxicity of pentachlorophenol is reduced, and the adsorption of the reproductive add Toxic than added directly to low, that is adsorption of carbon nanotubes decreased the concentration of free sodium pentachlorophenate, thereby reducing its reproductive toxicity. In summary, cytotoxicity test indexes are better than the individual level and individual level sensitive toxicity results there is a positive correlation. The cytotoxicity test is expected to replace the toxicity test the individual level has become a new fast, efficient and sensitive method of pollutants on soil biological toxicity.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X171.5

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