環保專案成果報告查詢系統

專案計畫編號	EPA-101-1605-02-01
經費年度	101
計畫經費	3,100 千元
專案開始日期	2012/01/19
專案結束日期	2012/12/31
專案主持人	闕斌如
主辦單位	環檢所
承辦人	許令宜
執行單位	國立中興大學
專案分類	環境監測及檢測
中文關鍵字	奈米金微粒，細胞毒性，生物標記
英文關鍵字	Gold nanoparticles, cytotoxicity, biomarkers
協同主持人	莊秀美
共同主持人
計畫聯絡人	闕斌如
計畫聯絡電話	04-22840896#120
計畫聯絡信箱	pjchueh@dragon.nchu.edu.tw

中文摘要	全球奈米科技正如火如荼的發展，雖然此尖端科技在各個領域上開拓新的希望、應用價值與新興產品，然而這些具有奈米結構的物質，其本身的化學性質與物理性質，會隨著粒徑大小的不同而有所變化，對人體的毒性危害或影響也可能有所改變，因此對於安全衛生環保亦將可能產生重大的衝擊。本研究團隊在去年度的計劃中建立以細胞連續及即時生長分析儀為奈米物質的細胞毒性之篩選平台，此項新的檢測方法，可以免除傳統細胞毒性測試方法之染劑或指示劑的使用，進而降低其與奈米物干擾所產生之不一致結果，並可發展更穩定、快速及可測試大量奈米微粒樣品的篩選平台。今年度的計劃延續以此細胞連續及即時生長分析儀為細胞毒性篩選平台，進行不同粒徑之奈米金溶液，對數種不同細胞株之細胞毒性動態影響，結果顯示奈米金溶液有效抑制細胞的生長，並呈現濃度相關性，但會因細胞株不同而有不同程度的抑制效果。以傳統的一般細胞毒性方法測試，如MTS，Trypan blue exclusion，Colony forming 等方法進行研究，也證實奈米金溶液有效抑制細胞的生長，而不同方法對奈米金溶液的細胞毒性有不同的靈敏度。計劃中也證實奈米金溶液可增加細胞凋亡或細胞週期停滯，進而抑制細胞的生長；本團隊也以 DNA microarray 分析找出幾個重要的生物分子標誌，這些特定蛋白分子明顯與基因損壞及細胞週期調控機制有重要的關係。本計劃以全面系統性的細胞毒性檢測，證實奈米金溶液以不同的機制抑制細胞的生長，歸納本計劃之執行成果如下:(1)本研究團隊針對多種哺乳類細胞株之生長特性，成功建立以細胞連續及即時生長分析儀分析奈米物質標毒性分析平台；(2)確定奈米金溶液在本系統的評估之下是有細胞毒性，且不同粒徑的奈米金對不同的細胞株有不同程度的生長抑制作用；(3) 一般細胞毒性分析系統較不易區分出不同細胞株對奈米金生長抑制作用之不同；(4)細胞連續及即時生長分析儀可適用於大多數的細胞株，包括無法利用其它細胞毒性方法測試的MRC-5細胞以及無法應用於colony-forming 及flow cytometry的PK-15細胞; (5)細胞連續及即時生長分析儀可在整個測試過程中，全面觀察細胞生長的動態變化，而非單一時間點的細胞毒性方法; (6)此方法並無一般傳統方法需要染劑的使用，因此並無染劑與奈米物質干擾的現象; (7)細胞電阻的測量為電腦自動化，減少以其他測試法所容易產生的人為操作誤差。研究中使用的奈米金為市售之純奈米金，實驗使用濃度為因應實驗需求直接強迫細胞暴露，與一般人體可能的暴露情況完全不同，目前無法以現有的研究結果反推人體暴露劑量與可能產生之立即毒性；因此本計劃的目的旨在建立細胞毒性評估工具與平台，而非依據有限之研究成果推論人體可能之暴露劑量與毒性反應。
中文關鍵字	奈米金微粒，細胞毒性，生物標記

中文摘要

全球奈米科技正如火如荼的發展，雖然此尖端科技在各個領域上開拓新的希望、應用價值與新興產品，然而這些具有奈米結構的物質，其本身的化學性質與物理性質，會隨著粒徑大小的不同而有所變化，對人體的毒性危害或影響也可能有所改變，因此對於安全衛生環保亦將可能產生重大的衝擊。本研究團隊在去年度的計劃中建立以細胞連續及即時生長分析儀為奈米物質的細胞毒性之篩選平台，此項新的檢測方法，可以免除傳統細胞毒性測試方法之染劑或指示劑的使用，進而降低其與奈米物干擾所產生之不一致結果，並可發展更穩定、快速及可測試大量奈米微粒樣品的篩選平台。今年度的計劃延續以此細胞連續及即時生長分析儀為細胞毒性篩選平台，進行不同粒徑之奈米金溶液，對數種不同細胞株之細胞毒性動態影響，結果顯示奈米金溶液有效抑制細胞的生長，並呈現濃度相關性，但會因細胞株不同而有不同程度的抑制效果。以傳統的一般細胞毒性方法測試，如MTS，Trypan blue exclusion，Colony forming 等方法進行研究，也證實奈米金溶液有效抑制細胞的生長，而不同方法對奈米金溶液的細胞毒性有不同的靈敏度。計劃中也證實奈米金溶液可增加細胞凋亡或細胞週期停滯，進而抑制細胞的生長；本團隊也以 DNA microarray 分析找出幾個重要的生物分子標誌，這些特定蛋白分子明顯與基因損壞及細胞週期調控機制有重要的關係。本計劃以全面系統性的細胞毒性檢測，證實奈米金溶液以不同的機制抑制細胞的生長，歸納本計劃之執行成果如下:(1)本研究團隊針對多種哺乳類細胞株之生長特性，成功建立以細胞連續及即時生長分析儀分析奈米物質標毒性分析平台；(2)確定奈米金溶液在本系統的評估之下是有細胞毒性，且不同粒徑的奈米金對不同的細胞株有不同程度的生長抑制作用；(3) 一般細胞毒性分析系統較不易區分出不同細胞株對奈米金生長抑制作用之不同；(4)細胞連續及即時生長分析儀可適用於大多數的細胞株，包括無法利用其它細胞毒性方法測試的MRC-5細胞以及無法應用於colony-forming 及flow cytometry的PK-15細胞; (5)細胞連續及即時生長分析儀可在整個測試過程中，全面觀察細胞生長的動態變化，而非單一時間點的細胞毒性方法; (6)此方法並無一般傳統方法需要染劑的使用，因此並無染劑與奈米物質干擾的現象; (7)細胞電阻的測量為電腦自動化，減少以其他測試法所容易產生的人為操作誤差。研究中使用的奈米金為市售之純奈米金，實驗使用濃度為因應實驗需求直接強迫細胞暴露，與一般人體可能的暴露情況完全不同，目前無法以現有的研究結果反推人體暴露劑量與可能產生之立即毒性；因此本計劃的目的旨在建立細胞毒性評估工具與平台，而非依據有限之研究成果推論人體可能之暴露劑量與毒性反應。

中文關鍵字

奈米金微粒，細胞毒性，生物標記

類型	檔名	檔案大小	說明	最新版公開日期
期末報告	epa-S0-1013652450-06-02[1].pdf	0MB		2013/2/1 上午 12:00:00

摘要	Nanomaterials have received considerable recent attention because of their unique properties and diverse applications in technology and life sciences. However, the nano-structures of these materials possess very different physical and chemical properties compared to their larger size counterparts. Given the increasing use of nanoparticles in a range of products, it has become crucial to develop a fundamental understanding of the biological effects of nanoparticle exposure. Many in vitro studies have suggested that nanoparticles are cytotoxic and genotoxic; however, results are inconsistent, mainly due to interference between nanoparticles and dye molecules commonly used in traditional cytotoxicity assays. The purpose of this study was to establish a systematic method for evaluating biological effects of nanogold utilizing the xCELLigence System, an electrical impedance-based, dye-free, real-time system, to continuously monitor the dynamic effects of nanogold on cell growth. The inhibitory effects of nanogold on the growth of numerous cell lines demonstrated using this system were also supported by several traditional in vitro toxicity assays, including MTS, trypan blue exclusion, colony forming assays. Mechanistic studies revealed that the action of nanogold is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context and concentration of nanogold. Additional evidence is provided from our DNA microarray analysis suggesting that many proteins involved in DNA damage responses and cell cycle regulation are significantly up-regulated or down-regulated. We suggest that the xCELLigence System is more effective and better applicable and the evidence is summarized as following: (1) We have established a screening platform utilizing the xCELLigence System to measure cytotoxicity of nanomaterials; (2) We have shown that gold nanoparticles possess different cytotoxicity levels toward several cell lines tested; (3) It is difficult to differentiate the subtle differences in cytotoxicity using traditional in vitro cytotoxicity assays; (4) the xCELLigence System is the most suitable assay for a wide-range cells, including MRC-5, which cannot be analyzed by MTS, trypan blue exclusion, and colony-forming ability; (5) the xCELLigence System provides a comprehensive representation of the entire duration of the assay that offers distinct and important advantages over traditional end-point assays; (6) there is no interference between nanomaterials and dye molecules in xCELLigence System; (7) cell impedances are monitored automatically after cells seeded onto E-plates for preventing from subsequent operation mistakes. The results presented in this study should provide helpful guidance on the future use of gold nanoparticles in occupational and medical applications.
英文關鍵字	Gold nanoparticles, cytotoxicity, biomarkers

摘要

Nanomaterials have received considerable recent attention because of their unique properties and diverse applications in technology and life sciences. However, the nano-structures of these materials possess very different physical and chemical properties compared to their larger size counterparts. Given the increasing use of nanoparticles in a range of products, it has become crucial to develop a fundamental understanding of the biological effects of nanoparticle exposure. Many in vitro studies have suggested that nanoparticles are cytotoxic and genotoxic; however, results are inconsistent, mainly due to interference between nanoparticles and dye molecules commonly used in traditional cytotoxicity assays. The purpose of this study was to establish a systematic method for evaluating biological effects of nanogold utilizing the xCELLigence System, an electrical impedance-based, dye-free, real-time system, to continuously monitor the dynamic effects of nanogold on cell growth. The inhibitory effects of nanogold on the growth of numerous cell lines demonstrated using this system were also supported by several traditional in vitro toxicity assays, including MTS, trypan blue exclusion, colony forming assays. Mechanistic studies revealed that the action of nanogold is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context and concentration of nanogold. Additional evidence is provided from our DNA microarray analysis suggesting that many proteins involved in DNA damage responses and cell cycle regulation are significantly up-regulated or down-regulated. We suggest that the xCELLigence System is more effective and better applicable and the evidence is summarized as following: (1) We have established a screening platform utilizing the xCELLigence System to measure cytotoxicity of nanomaterials; (2) We have shown that gold nanoparticles possess different cytotoxicity levels toward several cell lines tested; (3) It is difficult to differentiate the subtle differences in cytotoxicity using traditional in vitro cytotoxicity assays; (4) the xCELLigence System is the most suitable assay for a wide-range cells, including MRC-5, which cannot be analyzed by MTS, trypan blue exclusion, and colony-forming ability; (5) the xCELLigence System provides a comprehensive representation of the entire duration of the assay that offers distinct and important advantages over traditional end-point assays; (6) there is no interference between nanomaterials and dye molecules in xCELLigence System; (7) cell impedances are monitored automatically after cells seeded onto E-plates for preventing from subsequent operation mistakes. The results presented in this study should provide helpful guidance on the future use of gold nanoparticles in occupational and medical applications.

英文關鍵字

Gold nanoparticles, cytotoxicity, biomarkers