資料載入中.....
|
請使用永久網址來引用或連結此文件:
http://libir.tmu.edu.tw/handle/987654321/33759
|
| 題名: | 奈米粒子ZnO與TiO2之危害與保護 |
| 作者: | 劉怡伶 |
| 貢獻者: | 醫學科學研究所 |
| 日期: | 2010 |
| 上傳時間: | 2010-08-31 11:07:53 (UTC+8) |
| 摘要: | 中文摘要
隨著奈米科學與技術的蓬勃發展，奈米產品對於人類健康所造成的影響已受到很大的重視。以人類肝癌細胞與奈米粒子氧化鋅與二氧化鈦共同培養可評估奈米材料對細胞的毒性反應。為了降低奈米粒子的聚集特性與模擬原始狀態，奈米粒子氧化鋅包覆天然產品高分子天然葡萄聚醣deatran，並暴露於人類細胞已進行後續實驗。奈米氧化鋅在此實驗中被證實是有毒的，但是二氧化鈦則非。聯合兩項高科技影像成像系統：雷射共軛聚焦顯微鏡進行定位並且觀察細胞內胞器的變化，飛行式二次離子質譜儀則可提供奈米粒子的空間分佈並同時經由影像解析系統得到圖像的呈現。結合兩種影像系統，我們可以清楚的觀察細胞內胞器反映並同時可以看到離子分佈。經過以上初步實驗證實後，更了解為了讓奈米粒子的機轉，我們也希望接下來細胞經過奈米粒子氧化鋅的處理後，可以發生預期的細胞凋亡結果：自由基的產生、細胞色素C由去極化粒線體中釋放出來、caspase-3 水解酶的活化、到DNA斷裂都可被偵測出。更甚至，我們希望可以由西方點墨法證實細胞在經過奈米二氧化鈦暴露後的增生現象。初步的實驗中，我們獲得了細胞型狀改變與細胞經過奈米粒子暴露後的反應，但是以上實驗還需要其他相關技術來確認。因此，我們將會追蹤真實的細胞反應現象已獲取在奈米粒子下的生化機轉。此項實驗希望可以提高人類對於使用或是製造奈米材料的謹慎程度並且防止人類健康在高科技的環境下受到傷害。
Abstract
Given the intensive nano science and technological development, the health effect of nanosized products to human beings had been concerned. Nono-materials’ toxicological assessment on HepG2 cells were conducted after co-incubated with nano-sized ZnO and TiO2. Nanosized zinc oxide was coated with a natural product, dextran, to minimize its aggregate formation and appeared its original form while exposure to human beings. The ZnO particle-medium was cytotoxic but the TiO2 one was not. Associating with two high technical imaging systems, the laser confocal microscopy analysis pointed out the locations and condition of organelles in HepG2 cells after they treated with nanoparticles and then fluorescent dye; TOF-SIMS imaging offers a modality for simultaneously the spatial distribution of nanosparticles in cells. Combination of these two high technical images, it showed the effects to each organelle and the oxidative stress by nanoparticles and their distribution in HepG2 cells. To summarize all the primary data we had, we also expect the next steps that when cells were exposed to ZnO nanoparticles, a temporal pattern of apoptotic events was observed following the elevation of O2-, in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. Moreover, we expect to prove the cell proliferation phenomena by immunoblot assay after cells exposed to TiO2 nanoparticles. The primary data showed the morphology changes and also cells’ response, but it most still need to be identified by other techniques. After this, we will focus on tracing the really pathway or impacts from HepG2s to get more biochemical mechanism at this condition. This study highlighted the possibility for caution during the produce and purchase of nano-materials to prevent human health impacts. |
| 關聯: | 58頁 |
| 描述: | A、中文摘要…1
B、Abstract…2
C、The Content of Thesis Research…3
1. Specific Aim…3
2. Background…4
3. Flow chart…10
4. Materials and Methods…11
4. 1. Cell culture…11
4. 2. Preparation of Nanoparticles’ working medium…11
4. 3. Cell viability assays…12
4. 4. Cell cytotoxicity assays…13
4. 5. Cell apoptosis analysis…13
4. 6. Laser scanning confocal microscopy－Time-Lapse Imaging…14
4. 7. Time-of-flight secondary ion mass spectrometry
(TOF-SIMS) …14
4. 8. Morphological analysis TUNEL assay…15
4. 9. Immunoblot Analysis…16
4.10. Statistical analysis…16
5. Results…17
5.1. Measuring the sizes of nanoparticles…17
5.2. The effects of nanoparticles on cell viability and their cytotoxicity…20
5.3. ZnO particles induced apoptosis on HepG2 cells…24
5.4. Time-Lapse Imaging of cell damage…25
5.5. Internalization of NPs into HepG2 cells…33
5.6. Morphological analysis TUNEL assay…36
5.7. Identifying cell apoptosis and proliferation by
immunoblotting…38
5.8. The effects of nanoparticles on normal cells…39
6. Discussion…41
7. Reference…45
8. Acknowledgments…54
7. Reference
[1] Yokouchi, M., et al. Involvement of selective reactive oxygen species upstream of proapoptotic branches of unfolded protein response. J Biol Chem 283, 4252-4260 (2008).
[2] Buttke, T.M. & Sandstrom, P.A. Oxidative stress as a mediator of apoptosis. Immunology Today 15, 7-10 (1994).
[3] Warabi, E., et al. Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species. Free Radic Biol Med 42, 260-269 (2007).
[4] Gupta, S.C., et al. Chlorpyrifos induces apoptosis and DNA damage in Drosophila through generation of reactive oxygen species. Ecotoxicol Environ Saf (2010).
[5] Burbridge, D.J., Crampin, S., Viau, G. & Gordeev, S.N. Selected immobilization of individual nanoparticles by spot-exposure electron-beam-induced deposition. Nanotechnology 21, 045302 (2010).
[6] Pagan, I., Costa, D.L., McGee, J.K., Richards, J.H. & Dye, J.A. Metals mimic airway epithelial injury induced by in vitro exposure to Utah Valley ambient particulate matter extracts. J Toxicol Environ Health A 66, 1087-1112 (2003).
[7] Natusch, D.F., Wallace, J.R. & Evans, C.A., Jr. Toxic trace elements: preferential concentration in respirable particles. Science 183, 202-204 (1974).
[8] Kodavanti, U.P., et al. Variable pulmonary responses from exposure to concentrated ambient air particles in a rat model of bronchitis. Toxicol Sci 54, 441-451 (2000).
[9] Newman, M.D., Stotland, M. & Ellis, J.I. The safety of nanosized particles in titanium dioxide- and zinc oxide-based sunscreens. J Am Acad Dermatol 61, 685-692 (2009).
[10] Liu, S., Xu, L., Zhang, T., Ren, G. & Yang, Z. Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. Toxicology 267, 172-177 (2010).
[11] Oberdorster, G., Oberdorster, E. & Oberdorster, J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113, 823-839 (2005).
[12] Beckett, W.S., et al. Comparing inhaled ultrafine versus fine zinc oxide particles in healthy adults: a human inhalation study. Am J Respir Crit Care Med 171, 1129-1135 (2005).
[13] Chertok, B., et al. Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors. Biomaterials 29, 487-496 (2008).
[14] Nishiyama, N. & Kataoka, K. Medical applications of nanotechnology: polymeric micelles for drug delivery. Nippon Geka Gakkai zasshi. 106, 700-705 (2005).
[15] Lucas, R., et al. Synthesis and cellular uptake of superparamagnetic dextran-nanoparticles with porphyrinic motifs grafted by esterification. E-Polymers (2007).
[16] Lemarchand, C., Couvreur, P., Besnard, M., Costantini, D. & Gref, R. Novel polyester-polysaccharide nanoparticles. Pharmaceutical Research 20, 1284-1292 (2003).
[17] Bachelder, E.M., Beaudette, T.T., Broaders, K.E., Dashe, J. & Frechet, J.M. Acetal-derivatized dextran: an acid-responsive biodegradable material for therapeutic applications. J Am Chem Soc 130, 10494-10495 (2008).
[18] Wotschadlo, J., et al. Magnetic nanoparticles coated with carboxymethylated polysaccharide shells-Interaction with human cells. Journal of Magnetism and Magnetic Materials 321, 1469-1473 (2009).
[19] Tian, F., et al. A novel assay for the quantification of internalized nanoparticles in macrophages. Nanotoxicology 2, 232-242 (2008).
[20] Auffan, M., et al. CeO2 nanoparticles induce DNA damage towards human dermal fibroblasts in vitro. Nanotoxicology 3, 161-171 (2009).
[21] Pekkanen, J., Timonen, K.L., Ruuskanen, J., Reponen, A. & Mirme, A. Effects of ultrafine and fine particles in urban air on peak expiratory flow among children with asthmatic symptoms. Environ Res 74, 24-33 (1997).
[22] Penttinen, P., et al. Ultrafine particles in urban air and respiratory health among adult asthmatics. Eur Respir J 17, 428-435 (2001).
[23] Hall, S., Bradley, T., Moore, J.T., Kuykindall, T. & Minella, L. Acute and chronic toxicity of nano-scale TiO2 particles to freshwater fish, cladocerans, and green algae, and effects of organic and inorganic substrate on TiO2 toxicity. Nanotoxicology 3, 91-97 (2009).
[24] von Klot, S., et al. Increased asthma medication use in association with ambient fine and ultrafine particles. Eur Respir J 20, 691-702 (2002).
[25] Muhlfeld, C., Gehr, P. & Rothen-Rutishauser, B. Translocation and cellular entering mechanisms of nanoparticles in the respiratory tract. Swiss Med Wkly 138, 387-391 (2008).
[26] Mo, Y., Zhu, X., Hu, X., Tollerud, D.J. & Zhang, Q. Cytokine and NO release from peripheral blood neutrophils after exposure to metal nanoparticles: In vitro and ex vivo studies. Nanotoxicology 2, 79-87 (2008).
[27] Brown, J.S., Zeman, K.L. & Bennett, W.D. Ultrafine particle deposition and clearance in the healthy and obstructed lung. Am J Respir Crit Care Med 166, 1240-1247 (2002).
[28] Jaques, P.A. & Kim, C.S. Measurement of total lung deposition of inhaled ultrafine particles in healthy men and women. Inhal Toxicol 12, 715-731 (2000).
[29] Pekkanen, J., et al. Particulate air pollution and risk of ST-segment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air (ULTRA) study. Circulation 106, 933-938 (2002).
[30] Shah, A.P., et al. Effect of inhaled carbon ultrafine particles on reactive hyperemia in healthy human subjects. Environ Health Perspect 116, 375-380 (2008).
[31] Mao, Z., Wan, L., Hu, L., Ma, L. & Gao, C. Tat peptide mediated cellular uptake of SiO2 submicron particles. Colloids Surf B Biointerfaces 75, 432-440 (2010).
[32] Ma, L., et al. Oxidative stress in the brain of mice caused by translocated nanoparticulate TiO2 delivered to the abdominal cavity. Biomaterials 31, 99-105 (2010).
[33] Donaldson, K., et al. The pulmonary toxicology of ultrafine particles. J Aerosol Med 15, 213-220 (2002).
[34] Oberdorster, G., Oberdorster, E. & Oberdorster, J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113, 823-839 (2005).
[35] Huang, S., Chueh, P.J., Lin, Y.W., Shih, T.S. & Chuang, S.M. Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO2 long-term exposure. Toxicol Appl Pharmacol 241, 182-194 (2009).
[36] Ema, M., Kobayashi, N., Naya, M., Hanai, S. & Nakanishi, J. Reproductive and developmental toxicity studies of manufactured nanomaterials. Reprod Toxicol (2010).
[37] Rajagopalan, P., Wudl, F., Schinazi, R.F. & Boudinot, F.D. Pharmacokinetics of a water-soluble fullerene in rats. Antimicrob Agents Chemother 40, 2262-2265 (1996).
[38] Injac, R., et al. Acute doxorubicin pulmotoxicity in rats with malignant neoplasm is effectively treated with fullerenol C60(OH)24 through inhibition of oxidative stress. Pharmacol Rep 61, 335-342 (2009).
[39] Sayes, C.M., Marchione, A.A., Reed, K.L. & Warheit, D.B. Comparative pulmonary toxicity assessments of C60 water suspensions in rats: few differences in fullerene toxicity in vivo in contrast to in vitro profiles. Nano Lett 7, 2399-2406 (2007).
[40] Bermudez, E., et al. Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol Sci 77, 347-357 (2004).
[41] Colliver, T.L., et al. Atomic and molecular imaging at the single-cell level with TOF-SIMS. Anal Chem 69, 2225-2231 (1997).
[42] Stika, K.M., Bielat, K.L. & Morrison, G.H. Diffusible ion localization by ion microscopy: a comparison of chemically prepared and fast-frozen, freeze-dried, unfixed liver sections. J Microsc 118, 409-420 (1980).
[43] Lanekoff, I., et al. Time of Flight Mass Spectrometry Imaging of Samples Fractured In Situ with a Spring-Loaded Trap System. Anal Chem (2010).
[44] Chandra, S., Smith, D.R. & Morrison, G.H. Subcellular imaging by dynamic SIMS ion microscopy. Anal Chem 72, 104A-114A (2000).
[45] Strick, R., Strissel, P.L., Gavrilov, K. & Levi-Setti, R. Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes. J Cell Biol 155, 899-910 (2001).
[46] Severs, N.J. Freeze-fracture cytochemistry: a simplified guide and update on developments. J Microsc 161, 109-134 (1991).
[47] Cannon, D.M., Jr., Winograd, N. & Ewing, A.G. Quantitative chemical analysis of single cells. Annu Rev Biophys Biomol Struct 29, 239-263 (2000).
[48] Roddy, T.P., Cannon, D.M., Jr., Ostrowski, S.G., Ewing, A.G. & Winograd, N. Proton transfer in time-of-flight secondary ion mass spectrometry studies of frozen-hydrated dipalmitoylphosphatidylcholine. Anal Chem 75, 4087-4094 (2003).
[49] Clerc, J., Fourre, C. & Fragu, P. SIMS microscopy: methodology, problems and perspectives in mapping drugs and nuclear medicine compounds. Cell Biol Int 21, 619-633 (1997).
[50] McCandlish, C.A., McMahon, J.M. & Todd, P.J. Secondary ion images of the rodent brain. J Am Soc Mass Spectrom 11, 191-199 (2000).
[51] Roddy, T.P., Cannon, D.M., Jr., Meserole, C.A., Winograd, N. & Ewing, A.G. Imaging of freeze-fractured cells with in situ fluorescence and time-of-flight secondary ion mass spectrometry. Anal Chem 74, 4011-4019 (2002).
[52] Roddy, T.P., Cannon, D.M., Jr., Ostrowski, S.G., Winograd, N. & Ewing, A.G. Identification of cellular sections with imaging mass spectrometry following freeze fracture. Anal Chem 74, 4020-4026 (2002).
[53] Liebermann, T. & Knoll, W. Surface-plasmon field-enhanced fluorescence spectroscopy. Colloids and Surfaces A: Physicochemical and Engineering Aspects 171, 115-130 (2000).
[54] Wadu-Mesthrige, K., Xu, S., Amro, N.A. & Liu, G.Y. Fabrication and imaging of nanometer-sized protein patterns. Langmuir 15, 8580-8583 (1999).
[55] Utriainen, M., Leijala, A., Niinistö, L. & Matero, R. Chemical imaging of patterned inorganic thin-film structures by lateral force microscopy. Analytical Chemistry 71, 2452-2458 (1999).
[56] Yip, C.M., Brader, M.L., Frank, B.H., DeFelippis, M.R. & Ward, M.D. Structural studies of a crystalline insulin analog complex with protamine by atomic force microscopy. Biophys J 78, 466-473 (2000).
[57] Fulghum, J.E. Recent developments in high energy and spatial resolution analysis of polymers by XPS. Journal of Electron Spectroscopy and Related Phenomena 100, 331-355 (1999).
[58] Hagenhoff, B. High resolution surface analysis by TOF-SIMS. Mikrochimica Acta 132, 259-271 (2000).
[59] Nath, S., Kaittanis, C., Tinkham, A. & Perez, J.M. Dextran-coated gold nanoparticles for the assessment of antimicrobial susceptibility. Anal Chem 80, 1033-1038 (2008).
[60] Rejman, J., Oberle, V., Zuhorn, I.S. & Hoekstra, D. Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis. Biochemical Journal 377, 159-169 (2004).
[61] Lincz, L.F. Deciphering the apoptotic pathway: all roads lead to death. Immunol Cell Biol 76, 1-19 (1998).
[62] Maurer, B.J., Metelitsa, L.S., Seeger, R.C., Cabot, M.C. & Reynolds, C.P. Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)- retinamide in neuroblastoma cell lines. J Natl Cancer Inst 91, 1138-1146 (1999).
[63] Reed, D.J., Pascoe, G.A. & Thomas, C.E. Extracellular calcium effects on cell viability and thiol homeostasis. Environ Health Perspect 84, 113-120 (1990).
[64] Martinez, J. & Santibanez, J.F. Extracellular calcium modulates proliferation of factor dependent hemopoietic cells. Cell Biochemistry and Function 11, 101-105 (1993).
[65] Shahabuddin, M.S., Nambiar, M., Choudhary, B., Advirao, G.M. & Raghavan, S.C. A novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells. Invest New Drugs 28, 35-48 (2010).
[66] Galand, P. & Degraef, C. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell and Tissue Kinetics 22, 383-392 (1989).
[67] Nohynek, G.J., Lademann, J., Ribaud, C. & Roberts, M.S. Grey goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol 37, 251-277 (2007).
[68] Morganti, P. Use and potential of nanotechnology in cosmetic dermatology. Clinical, Cosmetic and Investigational Dermatology 3, 5-13 (2010).
[69] Beringer, J., et al. Nanolayers tackle a mega problem - Skin cancer: Effective, permanent UV-resistant textiles featuring nanolayers. Kettenwirk-Praxis, 41-43 (2007).
[70] Horie, M., et al. Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells. Chem Res Toxicol 22, 543-553 (2009).
[71] Karlsson, H.L., Cronholm, P., Gustafsson, J. & Möller, L. Copper oxide nanoparticles are highly toxic: A comparison between metal oxide nanoparticles and carbon nanotubes. Chemical Research in Toxicology 21, 1726-1732 (2008).
[72] Jeng, H.A. & Swanson, J. Toxicity of metal oxide nanoparticles in mammalian cells. J Environ Sci Health A Tox Hazard Subst Environ Eng 41, 2699-2711 (2006).
[73] Xiong, D.W., Fang, T., Chen, X.D., Sima, X.F. & Zhu, W.T. Oxidative stress effects and damage of nanoscale TiO2 and ZnO on zebrafish. Huan Jing Ke Xue 31, 1320-1327 (2010).
[74] Hu, C.W., et al. Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida. Soil Biology and Biochemistry 42, 586-591 (2010).
[75] Valant, J., et al. Hazardous potential of manufactured nanoparticles identified by in vivo assay. J Hazard Mater 171, 160-165 (2009).
[76] Kasemets, K., Ivask, A., Dubourguier, H.C. & Kahru, A. Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae. Toxicology in Vitro 23, 1116-1122 (2009).
[77] Wang, H., Wick, R.L. & Xing, B. Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans. Environ Pollut 157, 1171-1177 (2009).
[78] Hu, X., Cook, S., Wang, P. & Hwang, H.M. In vitro evaluation of cytotoxicity of engineered metal oxide nanoparticles. Sci Total Environ 407, 3070-3072 (2009).
[79] Aruoja, V., Dubourguier, H.C., Kasemets, K. & Kahru, A. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci Total Environ 407, 1461-1468 (2009).
[80] Liu, H.Y., et al. Effects of several nanooxides on the hatching rate of zebrafish embryos. Zhongguo Huanjing Kexue/China Environmental Science 29, 53-57 (2009).
[81] Zhu, X., et al. Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebrafish (Danio rerio) early developmental stage. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering 43, 278-284 (2008).
[82] Heinlaan, M., Ivask, A., Blinova, I., Dubourguier, H.C. & Kahru, A. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 71, 1308-1316 (2008).
[83] Palomaki, J., Karisola, P., Pylkkanen, L., Savolainen, K. & Alenius, H. Engineered nanomaterials cause cytotoxicity and activation on mouse antigen presenting cells. Toxicology 267, 125-131 (2010).
[84] Gerloff, K., Albrecht, C., Boots, A.W., Frster, I. & Schins, R.P.F. Cytotoxicity and oxidative DNA damage by nanoparticles in human intestinal Caco-2 cells. Nanotoxicology 3, 355-364 (2009). |
| 顯示於類別: | [醫學科學研究所] 博碩士論文
|
在TMUIR中所有的資料項目都受到原著作權保護.
|
