氧化鋅奈米微粒及其複合物之細胞毒性研究

Cytotoxicity study of zinc oxide hybrid nanoparticles

林睿昌(Ruey-Chang Lin)

指導教授 ： 鍾仁傑

國立臺北科技大學/工程學院/生物科技研究所/碩士(2012年)

摘要

目前奈米材料相關產品在市場上愈趨廣泛與精巧，奈米材料類似於次細胞結構增加與細胞反應性，甚至對環境及生物產生潛在的威脅，因此有必要深入探討奈米微粒與生物系統之交互作用。本實驗以氧化鋅奈米微粒 (ZnO)、氧化鋅結合二氧化矽 (ZnO/SiO2)、氧化鋅結合聚乙二醇 (ZnO/PEG)、氧化鋅結合二氧化矽並結合聚乙二醇 (ZnO/SiO2-PEG) 為材料，以 L929 細胞株 (L929 cell)進行實驗。H&E 與 DAPI 染色觀察細胞形態及細胞核變化；CCk-8 及 LDH Kit分析細胞毒性；流式細胞儀分析細胞週期、凋亡、壞死與攝取奈米微粒含量百分比；膠體電泳 (SDS PAGE) 檢視蛋白質變化。本研究所獲致結果如下：
一、 ZnO 於20 μg/ml劑量時產生許多空泡，40 μg/ml 細胞大量死亡呈現濃度依勢性質，ZnO-PEG，濃度大於 150 μg/ml 才具明顯毒性。
二、 ZnO/SiO2-PEG 奈米複合物，經由細胞主動攝取奈米微粒 (endocytosis) 於高濃度短時間與低濃度長時間，明顯呈現奈米微粒於細胞內部化 (internalization) 現象且具濃度與時間之依恃性質。
三、 細胞週期反應出奈米微粒使 DNA 受到傷害，S 期停滯現象，細胞需要多時間來修復受損之 DNA 以防止細胞產生突變。
四、 高濃度奈米微粒造成細胞凋亡與壞死變化，經由 Annexin V 與PI 螢光染色呈現在流式細胞儀偵測資料上。
五、 細胞回應奈米微粒之蛋白質變化，呈現在蛋白質電泳 (SDS PAGE) 圖上。
依據研究結果，可提供生醫檢測參考，奈米藥物設計新策略與治療系統應用資訊。

關鍵字

氧化鋅奈米微粒 ； 細胞毒性 ； 體外 ； 細胞染色 ； 細胞周期

並列摘要

In the present days, nanotech-based consume products are widespread and sophisticated. However, the size of nanoparticle is similar to the sub-cell structures so that it may lead to severe reactions with the cells which are potentially hazard to the environment and the organisms. It is urgent and necessary to deeply investigate the interactions between nanoparticle and cell. In this study, Zinc oxide nanoparticle (ZnO), zinc oxide/silicon dioxide composite nanoparticle (ZnO/SiO2), zinc oxide nanoparticle coated with polyethylene glycols (ZnO/PEG) and zinc oxide/silicon dioxide composite nanoparticle coated with polyethylene glycols (ZnO/SiO2-PEG) were used to study their interactions with biological system. L929 cell-line was cultured for the in vitro study. H&E staining and DAPI staining assays were carried out to observe the morphological changes of whole cell and nuclear; CCk-8 and LDH Kit analyses were used to study the cytotoxicity; quantification of cell cycle, apoptosis, necrosis, and cell uptake of nanoparticles were analyzed through the flow cytometer; and gel electrophoresis (SDS PAGE) was done to view the protein changes. The results including:
a. Under a 20 μg/ml dose of ZnO, the cells presented a number of vacuoles. Above 40 μg/ml, cell viability decreased dramatically in a dose-dependent manner. For ZnO-PEG, a concentration greater than 150 μg/ml leaded to apparent toxicity.
b. For ZnO/SiO2-PEG, the cells presented active endocytosis and the internalization of the nanoparticles were observed in a dose-dependent manner.
c. The cell cycle analyses showed that the nanoparticles leaded to damages of DNA, arrest of S phase and more time for restoring the damages.
d. High concentration of nanoparticle leaded to changes in the ratio of apoptosis and necrosis. Annexin V and PI fluorescent staining showed the differences through the flow cytometry analyses.
e. Through protein electrophoresis (SDS PAGE), the changes of protein in the cells responding to the nanoparticles were presented.

並列關鍵字

Zinc oxide composite nanoparticle ； Cytotoxicity ； In vitro ； Cell staining ； Cell cycle

參考文獻

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