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標題: | 以原子力顯微鏡探討奈米銀粒子經活性氧自由基造成人類角質細胞凋亡之現象 Silver nanoparticles induce ROS mediated apoptosis in human keratinocyte cells research by atomic force microscopy |
作者: | Jiou-Yi Lin 林久意 |
指導教授: | 彭福佐(Fu-Chuo Peng) |
共同指導教授: | 林世明(Shi-Ming Lin) |
關鍵字: | 奈米銀粒子,人類角質細胞,活性氧化物,細胞凋亡,原子力顯微鏡, Silver nanoparticle (AgNPs),HaCaT cell,reactive oxygen species (ROS),apoptosis,atomic force microscopy (AFM), |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 銀由於穩定並具有抑菌效果,千年來已用於銀器存放食物及酒,製作貨幣及器皿。許多研究顯示把銀奈米化後,奈米銀粒子(Silver nanoparticle,AgNPs)較銀具更佳的抗菌功能。先前研究指出AgNPs 會誘導細菌產生活性氧自由基(reactive oxygen species,ROS),ROS 會參與調節細胞凋亡過程,進而導致微生物的死亡。動物實驗顯示,奈米銀粒子會經由皮膚吸收造成發炎及水泡生成,然而奈米銀粒子對人類皮膚所造成的傷害尚未明瞭。現今AgNPs 廣泛被使用於商業產品中,增加了皮膚接觸AgNPs 的風險。因此,本研究的目的為探討AgNPs 對人類皮膚細胞所造成的影響。本研究選用10 nm 的AgNPs,處理於人類正常皮膚角質細胞(HaCaT),並以化學方式證實細胞發生凋亡現象,其後透過原子力顯微鏡(Atomic Force Microscope, AFM) 評估細胞型態的變化,AFM 目前已廣泛應用於生物樣品型態量測,並且可用於生物力學分析中。本實驗以細胞存活率試驗找出AgNPs 的半抑制濃度(concentration of 50 % inhibition, IC50) 於處理48 小時時約為5μg/ml。 接著以螢光顯微鏡及流式細胞儀觀察處理AgNPs 後細胞產生的ROS,並且於流式細胞儀及Hoechst 33342 染劑染色後觀察細胞發生凋亡現象。以上結果顯示HaCaT 細胞會因處理AgNPs 後經ROS 誘導而發生細胞凋亡。AFM 在觀察奈米材料及細胞的超微結構時為一項強大的工具,可適當的使用於測量細胞的生物物理特性。此外,AFM 在研究細胞凋亡的型態變化時可提供優良的影像。本研究在給予AgNPs 後造成HaCaT 細胞凋亡時,使用AFM
掃描後可見到細胞表面粗糙度及軟硬度增加,而黏滯力不變。綜合以上結果,本實驗以生化方式及物理方式證實AgNPs 會引起細胞死亡,是經由產生ROS 誘發細胞凋亡所致。 Owing to its stability and antimicrobial properties, silver has been used for thousands of years for food storage, currency coins and many other utensils. Numerous studies indicated that silver nanoparticles (AgNPs) have more antimicrobial properties than silver. Previous studies indicated that AgNPs could induce cellular apoptosis by producing reactive oxygen species (ROS). ROS regulated the process of apoptosis, which led to the destruction of microorganism. Animal studies reported that AgNPs could be absorbed through attaching to skin and caused inflammation and blisters. However, the damage of AgNPs on human skin has not been well understood. Yet recently, some commercial products containing AgNPs in the daily life increased the risk to exposure to AgNPs for our skin. Therefore, investigating the influence of AgNPs to human skin cell is the main propose of this research. In this study, human keratinocyte cell (HaCaT cells) was treated with AgNPs of size 10 nm in vitro, then confirmed the apoptosis phenomena by chemical methods, and finally evaluated the morphology changes by atomic force microscope (AFM). AFM had been widely used for biological scanning and force analysis, and ideal for depicting the surface morphology of our sample. In this study, MTT-based cell viability assays indicated an IC50 of approximately 5μg/ml of AgNPs after 48 hour exposure. AgNPs were found to be induce ROS generation in HaCaT cells by flow cytometry and fluoresce microscopy. AgNPs induced apoptosis in HaCaT cells by flow cytometry and IV Hoechst 33342 staining. Taken together, AgNPs induced cell death of HaCaT cells was via induction of ROS and apoptosis in biochemical feature. AFM has been shown to be a powerful tool for imaging materials at the nanometer level and for observing the ultrastructure of cells, which is appropriate for measuring the changes in the biophysical properties of the cells. Moreover, AFM offers an advantage over morphological characterization technique used to study apoptosis. After AgNPs treated with HaCaT cells in apoptosis phase, the surface roughness and stiffness were increased and adhesion was no significant changed. Over all, this study demonstrated that AgNPs induced cell death in HaCaT cell, which is likely to be induced by ROS, though which AgNPs trigger apoptosis using biochemical features and AFM. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62595 |
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顯示於系所單位: | 毒理學研究所 |
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