本文選題：二氧化鈦納米粒子 + 銅離子�。� 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究二氧化鈦納米粒子(TiO_2-NPs)與Cu~(2+)聯(lián)合,對牛血紅蛋白(Bovine Hemoglobin,BHb)、人支氣管上皮細(xì)胞(Human bronchial epithelial cells,16HBE)、斑馬魚胚胎的毒性作用,從分子、細(xì)胞和動物整體水平評價TiO_2-NPs與Cu~(2+)聯(lián)合產(chǎn)生的毒性效應(yīng)。方法:采用透射電鏡等表征TiO_2-NPs及其與Cu~(2+)混合物的表面形貌、粒徑及Zeta電位。以BHb為模型蛋白,采用紫外-可見吸收光譜法、熒光光譜法、同步熒光光譜法、時間分辨熒光光譜法及圓二色光譜法結(jié)合,探討TiO_2-NPs、Cu~(2+)及二者聯(lián)合與BHb的相互作用,并評價它們對BHb的毒性作用。以16HBE細(xì)胞為研究對象,采用噻唑藍(lán)(MTT)比色法測定TiO_2-NPs、Cu~(2+)及二者聯(lián)合對16HBE細(xì)胞的毒性作用,利用吖啶橙(AO)/溴化乙錠(EB)熒光雙染色法檢測16HBE細(xì)胞凋亡。選用斑馬魚胚胎為動物模型,以斑馬魚胚胎的孵化率、死亡率、畸形率及魚鰾正常發(fā)育率為指標(biāo),構(gòu)建體內(nèi)評價TiO_2-NPs、Cu~(2+)及二者聯(lián)合的毒性體系。結(jié)果:TiO_2-NPs粒徑約20 nm,Zeta電位為0.0606 mV,在水溶液中的平均粒徑為76.44nm。光譜學(xué)結(jié)果表明,TiO_2-NPs、Cu~(2+)及二者混合物與BHb均發(fā)生相互作用,均使BHb的熒光發(fā)生猝滅,其猝滅機(jī)制主要為靜態(tài)猝滅;TiO_2-NPs、Cu~(2+)及二者聯(lián)合均使BHb的構(gòu)象及二級結(jié)構(gòu)發(fā)生變化,螺旋結(jié)構(gòu)變得更為松散,三者對BHb均產(chǎn)生毒性效應(yīng)。TiO_2-NPs、Cu~(2+)及二者聯(lián)合對16HBE細(xì)胞的毒性作用均呈劑量效應(yīng)、時間效應(yīng);AO/EB結(jié)果證明TiO_2-NPs、Cu~(2+)及二者聯(lián)合均可以誘導(dǎo)16HBE細(xì)胞凋亡;0-800mg·L-1的TiO_2-NPs聯(lián)合10 mg·L-1的Cu~(2+)對16HBE細(xì)胞的毒性作用強(qiáng)于TiO_2-NPs。當(dāng)TiO_2-NPs濃度介于0-200 mg·L-1時,斑馬魚胚胎死亡率均小于15%,畸形率、孵化率及魚鰾正常發(fā)育率與對照組均無顯著性差異;當(dāng)Cu~(2+)濃度大于0.125 mg·L-1時,斑馬魚胚胎72 h孵化率(26%)和染毒終點(diǎn)時魚鰾正常發(fā)育率(27%)較低、染毒終點(diǎn)時的死亡率(8%)和畸形率(25%)較高,且存在劑量依賴關(guān)系;0-1mg·L-1 TiO_2-NPs與0.125 mg·L-1 Cu~(2+)聯(lián)合對斑馬魚胚胎的毒性作用與單獨(dú)Cu~(2+)相近,當(dāng)TiO_2-NPs的濃度為5-200 mg·L-1時,二者聯(lián)合后斑馬魚胚胎的孵化率和魚鰾正常發(fā)育率均增高,死亡率和畸形率均降低。結(jié)論:TiO_2-NPs、Cu~(2+)及二者聯(lián)合均對BHb具有一定的毒性作用。體外0-800 mg·L-1的TiO_2-NPs聯(lián)合10 mg·L-1 Cu~(2+)對16HBE細(xì)胞呈增毒效應(yīng)。體內(nèi)5-200 mg·L-1的TiO_2-NPs與0.125 mg·L-1的Cu~(2+)聯(lián)合對斑馬魚胚胎呈減毒效應(yīng)。
[Abstract]:Objective: to study the toxic effects of TIO _ 2 nanoparticles (TIO _ 2-NPs) combined with Cu2 on bovine hemoglobinine (BHB), human bronchial epithelial cells (16HBE) and zebrafish embryos. The toxicity of TiO- 2-NPs combined with Cu2 was evaluated at the cellular and animal levels. Methods: the surface morphology, particle size and Zeta potential of TiO2-NPs and their mixtures with Cu2 were characterized by transmission electron microscopy. Using BHB as model protein, the interaction of TiO2-NPsCu2 and its combination with BHB was investigated by UV-Vis absorption spectrum, fluorescence spectrum, synchronous fluorescence spectrum, time-resolved fluorescence spectrum and circular dichroism spectroscopy. Their toxicity to BHB was evaluated. The cytotoxicity of TiO2-NPsCu2 and its combination on 16HBE cells was determined by MTT colorimetry. The apoptosis of 16HBE cells was detected by acridine orange (AO) / ethidium bromide (EB) fluorescence double staining. Using zebrafish embryos as animal model, the toxicity system for evaluating TiO2-NPsCu ~ (2) and its combination in vivo was constructed by using the hatching rate, mortality rate, deformity rate and normal development rate of swim bladder as the indexes of zebrafish embryos. Results the Zeta potential of 20 nmPs was 0.0606 MV and the average particle size in aqueous solution was 76.44 nm. The spectroscopic results show that TiO2-NPsCu ~ (2) and their mixtures interact with BHB, and both of them cause fluorescence quenching of BHB. The quenching mechanism is mainly static quenching of TiO2-NPsOCu2 and the combination of both of them to change the conformation and secondary structure of BHB. The spiral structure became looser, and the toxic effects of the three on BHB. The toxicity of TiO- 2-NPsCu ~ (2) and their combination on 16HBE cells were dose-dependent. The results of AOP / EB showed that TiO2-NPsCu ~ (2) and its combination could induce apoptosis of 16HBE cells. The toxicity of TiO2-NPs combined with Cu2 (10 mg L-1) on 16HBE cells was stronger than that of TiO2-NPs. When tio _ 2-NPs concentration was between 0-200 mg 路L ~ (-1), the embryonic mortality of zebrafish was lower than 15 mg 路L ~ (-1), and there was no significant difference in abnormal rate, hatching rate and normal development rate of swim bladder between the two groups, and when the concentration of Cu2 was more than 0.125 mg 路L ~ (-1), there was no significant difference between the two groups. The hatching rate of zebrafish embryos at 72 h (26%) and the normal development rate of swim bladder at the end of exposure (27%) were lower, the mortality rate (8%) and abnormal rate (25%) were higher at the end point of exposure to zebrafish. The toxicity of TiO2-NPs and 0.125 mg L-1 Cu2 to zebrafish embryos was similar to that of Cu2 alone. When the concentration of TiO2-NPs was 5-200mg L-1, the hatching rate of zebrafish embryos and the normal development rate of swim bladder increased after the combination of TiO2-NPs and TiO2-NPs. Mortality and deformity rate decreased. Conclusion\% TiO2-NPsCu ~ (2) and their combination have some toxic effects on BHB. In vitro, 0 800 mg L -1 TiOs 2 NPs combined with 10 mg L -1 Cu2 showed a toxic effect on 16 HBE cells. The combination of TiOs 2-NPs 5-200mg L-1 and Cu2 (0.125 mg L-1) showed a attenuated effect on zebrafish embryos.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R99

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