本文選題：碳納米管　切入點(diǎn)：五氯酚鈉　出處：《南京大學(xué)》2015年碩士論文

【摘要】：碳納米管(Carbonnanotubes,CNTs)自1991年被發(fā)現(xiàn)以來,以其獨(dú)特的理化性質(zhì)和較好的生物相容性被廣泛研究和利用。與此同時,CNTs也大量進(jìn)入環(huán)境中,對生態(tài)系統(tǒng)造成的影響日益增加,其毒性研究也逐漸受到重視。五氯酚(pentachlorophenol,PCP)及其鈉鹽作為一種除莠劑和殺蟲劑,在上世紀(jì)九十年代之前被大量使用,雖已停產(chǎn)并禁用,但由于其在環(huán)境中不易降解,目前還有大量PCP殘留在環(huán)境中,有機(jī)污染物PCP與大分子材料CNTs復(fù)合后又會產(chǎn)生新的毒性效應(yīng)。赤子愛勝蚓(Eisenia foetida)作為土壤生態(tài)系統(tǒng)中的標(biāo)準(zhǔn)受試生物,已有CNTs對其毒性的研究,但蚯蚓在個體水平上的毒理學(xué)試驗(yàn)耗時長,實(shí)驗(yàn)動物用量大,且靈敏度不高,其實(shí)際應(yīng)用非常有限。而細(xì)胞試驗(yàn)耗材少、速度快、靈敏度高,且細(xì)胞的毒性指標(biāo)與個體毒性指標(biāo)又有一定的相關(guān)性,可在很大程度上反應(yīng)個體水平的毒性。本研究對探索了赤子愛勝蚓體腔細(xì)胞的體外培養(yǎng)方法,并通過體外染毒,研究了 CNTs和PCP-Na單獨(dú)或聯(lián)合作用下對蚯蚓細(xì)胞的急性毒性、氧化損傷、基因損傷和膜結(jié)構(gòu)損傷的影響。同時研究了 CNTs和PCP-Na對赤子愛勝蚓的生殖毒性試驗(yàn),從多層面探討兩種受試物的毒性機(jī)理。本研究主要結(jié)果如下:(1)赤子愛勝蚓體腔細(xì)胞在體外可以懸浮狀態(tài)進(jìn)行原代培養(yǎng)。用96孔板培養(yǎng)時,最佳培養(yǎng)條件為溫度25℃、培養(yǎng)基為RPMI-1640培養(yǎng)基、不通CO2、細(xì)胞濃度為2-4×104個/孔(1-2×105個/mL),培養(yǎng)時間為24h。細(xì)胞存活率可達(dá)100%。(2)赤子愛勝蚓體腔細(xì)胞體外培養(yǎng)染毒,PCP-Na的LC50為104.46mg/L,其毒性介于蚯蚓個體人工土壤試驗(yàn)染毒第7天和第14天急性毒性之間。而與濃度為50mg/L的CNTs復(fù)合染毒試驗(yàn)中,直接添加復(fù)合染毒LC50為109.39 mg/L,CNTs吸附PCP-Na后染毒LC50為112.92 mg/L,兩種方法復(fù)合染毒后急性毒性都略有減小。碳納米管急性毒性非常小,試驗(yàn)中未測得其LC50值,這與個體水平上的急性毒性試驗(yàn)結(jié)果一致。(3)碳納米管與五氯酚鈉都對細(xì)胞產(chǎn)生氧化損傷,產(chǎn)生ROS,并能引起脂質(zhì)過氧化,生成MDA。低脅迫下,細(xì)胞能作出應(yīng)激反應(yīng),SOD活性增大,高脅迫下,SOD活性降低。CAT是最不敏感的生物標(biāo)志物。這與個體水平上研究結(jié)果類似。(4)用彗星試驗(yàn)評價蚯蚓受兩種受試物的脅迫作用,較為靈敏。試驗(yàn)結(jié)果顯示,各染毒組與對照組相比均有顯著性差異,碳納米管單獨(dú)染毒時,其與DNA損傷有明顯的劑量-效應(yīng)關(guān)系。而五氯酚鈉單獨(dú)染毒時,濃度較高的染毒組DNA損傷反而較小。CNTs與PCP-Na兩者復(fù)合染毒時,其遺傳毒性大于受試物單一染毒時的毒性,表現(xiàn)出一定的協(xié)同作用。試驗(yàn)結(jié)果與個體水平上彗星試驗(yàn)的結(jié)果類似。(5)高濃度五氯酚鈉、50mg/L碳納米管組以及復(fù)合染毒組均能對細(xì)胞的膜結(jié)構(gòu)造成損傷,增加細(xì)胞膜的通透性,并降低細(xì)胞的線粒體跨膜電位(Mitochondrial Membrane Potential,MMP)。五氯酚鈉對膜的損傷比較大,兩者復(fù)合染毒后,對細(xì)胞膜的毒性降低,兩種污染物呈拮抗作用。受試物對細(xì)胞膜結(jié)構(gòu)的損傷程度與MDA含量有一定的相差性,表明對膜結(jié)構(gòu)的損傷主要是氧化損傷。(6)個體水平上生殖試驗(yàn)結(jié)果表明,游離態(tài)的碳納米管可能會對蚯蚓生長起到一定的促進(jìn)作用。高濃度的五氯酚鈉具有較強(qiáng)的生殖毒性,會明顯減少蚯蚓蚓繭的產(chǎn)量,低濃度的五氯酚鈉對蚯蚓生長的影響較大,但做為補(bǔ)償,蚯蚓的平均產(chǎn)繭量有所提高。碳納米管對蚯蚓的生殖毒性較低,其與五氯酚鈉復(fù)合染毒時,五氯酚鈉的生殖毒性也降低,且吸附后添加的生殖毒性比直接添加的要低,表明是碳納米管的吸附作用降低了游離態(tài)五氯酚鈉的濃度,從而降低其生殖毒性。綜上所述,細(xì)胞毒性試驗(yàn)各指標(biāo)都要比個體水平指標(biāo)靈敏,且與個體水平毒性測定結(jié)果有一定的正相關(guān)性。細(xì)胞毒性試驗(yàn)有望取代蚯蚓個體水平的毒性試驗(yàn)成為新的快速、高效、靈敏的研究污染物對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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X171.5

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