以原子力顯微鏡探討奈米氧化鋅粒子經活性氧自由基造成人類角質細胞凋亡之現象

Abstract

氧化鋅(ZnO)是一種白色粉末，不溶於水，它被廣泛用作許多材料和產品中的添加劑，包括塑料、塗料、食品等等。在材料科學中的應用，氧化鋅具有高折射率，高導熱性，抗菌，防紫外線的性能及功效，因為ZnO具有可反射阻隔紫外線的效果，現今為人廣泛使用在防曬用產品。隨著奈米科技的逐步發展，奈米粒子之材料已應用於日常生活中，如防曬用產品也將ZnO奈米化為更能達到防曬效果。然而，有研究指出奈米級顆粒可以進入體內影響人類細胞，如人類肝細胞 （human liver cells, HepG2）。由於防曬產品首先接觸是皮膚，而文獻尚未詳細報導氧化鋅奈米粒子 （Zinc oxide nanoparticles, ZnO NPs） 對皮膚角質細胞的影響，因此本研究選用人類角質細胞（human keratinocyte cell, HaCaT cell） 來探討皮膚暴露於ZnO NPs後產生細胞毒性、氧化壓力以及細胞凋亡的傷害。細胞存活率顯示HaCaT cell暴露24小時於半抑制濃度(Concentration of 50 % inhibition, IC50) 50μg/ml的ZnO NPs，藉由流式細胞儀和螢光顯微鏡發現ZnO NPs對HaCaT cells 會產生活性氧化物（reactive oxygen species, ROS）。另外，以Annexin V 染劑分析細胞凋亡及 PI 染劑分析細胞壞死，結果顯示HaCaT細胞在18小時產生凋亡反應、24小時產生壞死現象，除此之外，以偵測細胞凋亡的 Hoechst 33342 染劑也顯現處理50 μg/ml ZnO NPs 18小時後產生凋亡反應。原子力顯微鏡(Atomic force microscopy, AFM ) 是一個高解析度的探針掃描顯微鏡，對於觀察細胞上奈米級的影象微結構是相當強大的工具，並且相當適合以物理特性測量細胞上的改變，所以AFM提供了用來研究細胞凋亡形態學特徵的技術優勢。HaCaT 細胞經由處理了ZnO NPs，細胞表面的粗糙度增加、硬度減少、黏滯力無明顯改變。綜合以上結果，本研究以生化與生物物理特徵之實驗數據證實ZnO NPs引發HaCaT 細胞死亡是藉由誘導ROS和細胞凋亡。

Zinc oxide (ZnO) is a white powder that is insoluble in water, which is widely used as an additive in numerous materials and products including plastics, paints, foods. For material science applications, zinc oxide has high refractive index, antibacterial and UV-protection properties. ZnO are used in various products, including cosmetics and sunscreens due to UV-filtering properties. The production of engineered nanoparticles is growing rapidly as the field of nanotechnology continues to expand. Modern sunscreen contain insoluble ZnO nanoparticle (ZnO NPs), which is reflect ultraviolet (UV) more efficiently than larger particles. However, cosmetic research suggest that vesicle materials may enter human cell, like human liver cells (HepG2). Sunscreen products is the first contact with the skin, and the underlying mechanisms of zinc oxide adverse effects have not been fully characterized. This study was designed to investigate the cytotoxicity, oxidative stress and apoptosis by ZnO nanoparticles in human keratinocyte cell (HaCaT cell). Cell viability assays indicated an IC50 approximately 50 μg/ml of ZnO nanoparticles after 24 h of exposure. ZnO nanoparticles were found to induce reactive oxygen species (ROS) generation in HaCaT cells by flowcytometry and fluoresce microscopy. ZnO nanoparticles were also investigated using Annexin V staining for apoptosis analysis and P2 staining for necrosis analysis. ZnO nanoparticles displaced apoptosis at 18 hr and then necrosis at 24 hr. Additional, Hoechst 33342 staining revealed apoptosis in HaCaT cells after 50 μg/ml ZnO nanoparticles 18hr treatment. Atomic force microscopy (AFM) is a very high resolution type of scanning probe microscopy. It has been shown to be a powerful tool for imaging materials at the nanometer level and for observing the ultrastructure of a cell. This method is appropriate for measuring the change in the biophysical properties of the cell. AFM offers an advantages over morphological characterization technique used to study apoptosis. After ZnO nanoparticles treatment the HaCaT cells, the surface roughness was increased, the stiffness was decreased and the adhesion was no significant changed. In conclusion, this study demonstrated that ZnO nanoparticles induced cell death of HaCaT cells was via induction of ROS and apoptosis by biochemical feature and AFM.