二氧化鈦奈米粒子所造成人類細胞毒性與基因毒性之探討

Study the Cytotoxicity and Genotoxicity of TiO2 Nanoparticles in Human Cells

江蘇峰(Sufeng Chiang)

指導教授 ： 莊曜宇

國立臺灣大學/電機資訊學院/電機工程學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10110

摘要

隨著奈米科技的快速發展，奈米材料已廣泛應用於各個層面。其中以二氧化鈦奈米粒子 (titanium dioxide nanoparticles) 用途最普遍也最為常見。因為其尺寸大小為奈米等級，奈米材料具有和塊材 (bulk material) 截然不同的物理與化學性質。這些特質使奈米材料有很好的發展與應用，卻也可能因此對人體健康造成不良影響。根據過去文獻，已知二氧化鈦奈米粒子可造成細胞損壞，導致細胞凋亡 (apoptosis)，並引起發炎反應。本研究以人類淋巴球母細胞 (TK6) 與人類肺部上皮細胞 (A549) 針對最廣為使用的二氧化鈦奈米粒子，探討其所可能引起的細胞毒性 (cytotoxicity) 與基因毒性 (genotoxicity)。希望在奈米科技發展的同時，能提供奈米材料在生物毒性方面的相關資訊，使奈米科技得以更安全地發展。本研究採用40 nm的銳鈦礦 (anatase) 二氧化鈦奈米粒子處理人類細胞株，經過24小時後，利用Colony formation assay與MTT assay觀察奈米粒子暴露所造成細胞毒性。基因晶片 (DNA microarray) 主要應用在觀察大量基因之表現，為找出特定基因調控極為方便、快速與可靠的方法。本研究同時利用基因晶片觀察二氧化鈦奈米粒子所造成基因毒性。希望能找出奈米粒子所誘發之特定基因，以建立二氧化鈦奈米粒子造成基因毒性之生物指標 (biomarker)，並進而研究二氧化鈦奈米粒子對人體細胞的生理機制之影響。實驗結果顯示，二氧化鈦奈米粒子暴露所造成人類淋巴球母細胞與人類肺部上皮細胞之細胞毒性，需要較長時間才會表現，在短期內並無法觀察到任何細胞毒性。二氧化鈦奈米粒子暴露所造成人類淋巴球母細胞與人類肺部上皮細胞之基因毒性經過基因晶片分析發現，二氧化鈦奈米粒子引發細胞表現大量細胞修復基因與細胞凋亡相關基因，同時亦觀察到細胞內調控氧化壓力 (oxidative stress) 平衡與發炎反應 (inflammatory response) 相關基因的表現。此結果顯示二氧化鈦奈米粒子造成細胞氧化壓力增加，並進而造成細胞傷害，最後還可能引起發炎反應。本研究所得結果，仍有待進一步的研究與探討。另一方面，經過觀察二氧化鈦奈米粒子粒徑與外觀型態，本研究發現二氧化鈦奈米粒子在水溶液中會以極快速度聚結 (aggregate) 成較大粒徑粒子。此特性導致傳統的活體與細胞實驗方法難以觀察到奈米粒子所可能引發的毒性影響，因此對於傳統實驗方法，仍需經過檢討與修正，才能實際應用於奈米粒子的毒性測試。

關鍵字

二氧化鈦奈米粒子 ； 細胞毒性 ； 基因毒性 ； 基因晶片 ； 生物指標 ； 氧化壓力 ； 發炎反應

並列摘要

Along with rapid development of nanotechnology, extensive applications of nanomaterials have been carried out in diverse aspects. Among them, titanium dioxide nanoparticles provide most widespread applications and they are the most common nanomaterials. Because of the nano scale, nanomaterials are substantially different in specific physical and chemical properties from those bulk materials of the same composition. These characteristics allow excellent development and application of nanomaterials; yet the adverse effects on human health may also be generated possibly. According to past literatures, it has been known that titanium dioxide nanoparticles could cause cell damage, apoptosis and inflammatory response. In this study, a human lymphoblastoid cell line (TK6), and a human pulmonary epithelial cell line (A549) were used to determine possible cytotoxicity and genotoxicity induced by titanium dioxide nanoparticles, which have been used most extensively. It was expected to provide relevant information about biohazard of nanomaterials and to facilitate safer development of nanotechnology. In this study, human cell lines were exposed to titanium dioxide nanoparticles of 40 nm in anatase. After 24 hours, Colony formation assay and MTT assay were used to assess cytotoxicity derived from exposure of titanium dioxide nanoparticles. DNA microarray is mainly applied as a high-throughput analysis for expression levels of a large quantity of genes, which allocates a very convenient, fast and reliable method to find out the mechanism of specific genes. At the same time, DNA microarray was used to analyze the genotoxicity induced by titanium dioxide nanoparticles. It was expected to determine specific genes induced by nanoparticles, so as to find out detectable biomarkers of the genotoxicity induced by titanium dioxide nanoparticles, and to further study on influence of titanium dioxide nanoparticles towards physiological mechanism of human cells. The results showed that the cytotoxicity of human lymphoblastoid cell line and human pulmonary epithelial cell line derived from exposure of titanium dioxide nanoparticles took longer period of time to appear, and none of the cytotoxicity was shown within short period of time. As for the genotoxicity of human lymphoblastoid cell line and human pulmonary epithelial cell line induced by titanium dioxide nanoparticles, after analysis with DNA microarray, it was found out that titanium dioxide nanoparticles triggered expression of massive amount of cell repair genes and apoptosis-related genes. The expression of some genes related to balance of oxidative stress and inflammatory response in cells were also observed at the same time. The results showed that exposure of titanium dioxide nanoparticles resulted in increase of oxidative stress of cells and further damage to cells, which may even induced inflammatory response at the end. The results obtained from this study still need to be further researched and discussed. On the other hand, after observation on size and appearance of titanium dioxide nanoparticles, it was discovered that the titanium dioxide nanoparticles would aggregate into larger particles in an extremely rapid manner. Such characteristics are obstacles for traditional method of animal and cell experiments on assessing possible toxic influence induced by nanoparticles. Therefore, the traditional methods of experiments still need to be reviewed and modified, so they can be actually applicable to toxicity test of nanoparticles.

並列關鍵字

titanium dioxide nanoparticle ； cytotoxicity ； genotoxicity ； DNA microarray ； biomarker ； oxidative stress ； inflammatory response

參考文獻

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