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研究生:侯瑞良
研究生(外文):Jui-Liang Hou
論文名稱:單層奈米碳管誘發人類肺細胞凋亡的訊息傳遞路徑
論文名稱(外文):The apoptotic signal transduction pathways induced by single-wallcarbon nanotube in human lung cells
指導教授:趙瑞益趙瑞益引用關係邱鐵雄邱鐵雄引用關係
指導教授(外文):Jui-I ChaoChiu,TED H
學位類別:碩士
校院名稱:慈濟大學
系所名稱:藥理暨毒理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:53
中文關鍵詞:單層奈米碳管
外文關鍵詞:single-wallcarbon nanotube
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中文摘要
奈米碳管是一種含碳的奈米材料,近幾年來被大量地開發作為生物醫學的應用,然而,奈米碳管對人類細胞毒性的影響仍不清楚,因此本論文利用人類肺細胞模式,研究單層奈米碳管誘發細胞毒性的可能機制。我們發現單層奈米碳管以十二烷基硫酸鈉或水當溶劑時,對人類A549肺上皮細胞及HFL-1肺纖維母細胞,皆會誘發細胞毒性,並且40-100 μg/mL單層奈米碳管會誘發caspase-3蛋白的活化及造成細胞凋亡。同時,我們發現單層奈米碳管會誘發ERK及p38有絲抗原活化蛋白激酶磷酸化的增加;以PD98059 (一種ERK專一抑制劑)或SB202190 (一種p38專一抑制劑)分別阻斷A549細胞的ERK和p38路徑時,皆會降低單層奈米碳管的細胞毒性。再者,單層奈米碳管會誘發p53磷酸化蛋白量的增加,以pifithrin-α (一種p53專一抑制劑)處理時,則會降低單層奈米碳管所造成的細胞死亡。有趣地,我們發現單層奈米碳管也會誘發Daxx蛋白的表達,當阻斷ERK、p38及p53路徑時,則會降低單層奈米碳管所誘發Daxx蛋白表現量。綜合以上的結果,我們推測單層奈米碳管,可經由活化ERK、p38及p53參與調控人類肺細胞的凋亡及Daxx蛋白的表達。
Abstract
Single-walled carbon nanotube (SWCNT) is a carbon derivative nanomaterial that has been extensively developed for biomedical applications. However, the cytotoxic effects of SWCNT toward human cells are still unclear. In this study, we investigated the cytotoxicity of SWCNT and its possible mechanisms using a human lung cell model. We found that SWCNTs, which were dissolved in sodium dodecyl sulfate or water, induced cytotoxicity in both A549 lung epithelial cells and HFL-1 lung fibroblasts. Moreover, treatment with 40-100 μg/mL SWCNTs induced the active caspase-3 protein expression and apoptosis. Meanwhile, the phosphorylations of ERK, p38, and p53 were increased by SWCNTs. Co-treatment with SB202190 (a specific p38 kinase inhibitor) or PD98059 (a specific ERK inhibitor) reduced the SWCNT-induced cytotoxicity in A549 cells. Furthermore, treatment with pifithrin-α (a specific p53 inhibitor) reduced the SWCNT-induced cell death. Interestingly, SWCNTs induced the expression of Daxx protein. The blockade of ERK, p38, and p53 pathways decreased the Daxx protein expression in the SWCNT-treated cells. Taken together, we suggest that SWCNT induces the phosphorylations of ERK, p38, and p53, which may induce apoptosis and regulate the Daxx protein expression in the human lung cells.
目錄 頁
目錄…………………………………………………………………………………..Ⅰ
圖目錄………………………………………………………………………………..Ⅲ
中英文專有名詞對照表……………………………………………………………..Ⅳ
中文摘要……………………………………………………………………………..Ⅴ
英文摘要……………………………………………………………………………..Ⅵ
壹、文獻回顧…………………………………………………………………………1
一、奈米材料在生物醫學的應用…………………………………………………1
二、奈米碳管的合成............................................................................................... 1
三、奈米碳管在生物醫學的應用…………………………………………………2
四、奈米碳管的毒性………………………………………………………………3
五、細胞凋亡的調控………………………………………………………………4
六、奈米顆粒與p38訊息傳遞路徑的活化的關係............................................... 5
七、奈米顆粒與ERK訊息傳遞路徑的活化的關係................................................6
八、奈米顆粒與p53訊息傳遞路徑的活化的關係............................................... 6
九、Daxx的細胞凋亡路徑……………………………………………………… 7
十、研究動機………………………………………………………………………7
貳、材料與方法…………….………………………………………………………… 8
一、試劑與抗體……………………………………………………………………8
二、單層奈米碳管的溶液製備……………………………………………………8
三、穿透式電子顯微鏡(Transmission electron microscope)…………………… 8
四、細胞培養(Cell culture) ……………………………………………………… 8
五、細胞毒性分析(Cytotoxicity assay) ………………………………………… 9
六、細胞凋亡分析(Apoptosis assay) ……………………………………………10
七、西方墨點法(Western blot) ………………………………………………… 10
八、免疫螢光分析(Immunofluorescence analysis)……….……………………...11
II
九、統計與數據分析(Statistics and data analysis) ……………………………...12
參、結果………………………………………………………………………………13
一、單層奈米碳管在不同溶劑下之形態……………………………………… 13
二、單層奈米碳管誘發人類肺細胞的毒性…………………………………… 13
三、單層奈米碳管誘發caspase-3的活化及細胞凋亡………………………… 13
四、單層奈米碳管誘發p38及ERK的蛋白磷酸化增加………………………....14
五、單層奈米碳管誘發細胞內磷酸化p53蛋白表達增加……………………....14
六、抑制p38及ERK的活化會降低單層奈米碳管所誘發的細胞毒性…………14
七、抑制p53的磷酸化表現會降低單層奈米碳管所誘發的細胞毒性…………15
八、單層奈米碳管誘發細胞內Daxx蛋白的表達增加………………………… 15
九、抑制p38及ERK的活化會降低單層奈米碳管所誘發Daxx蛋白的表達…...15
十、抑制p53的表現會降低單層奈米碳管所誘發Daxx蛋白的表達…………...16
肆、討論………………………………………………………………………………17
一、不同溶劑下對奈米碳管誘發肺細胞毒性的影響………………………… 17
二、單層奈米碳管誘發ERK及p38蛋白磷酸化路徑……………………………17
三、單層奈米碳管誘發p53蛋白磷酸化路徑……………………………………18
四、單層奈米碳管誘發Daxx蛋白路徑…………………………………………18
五、總結………………………………………………………………………… 19
伍、參考文獻……………………………………………………………………........20
III
圖目錄 頁
圖一、單層奈米碳管在穿透式電子顯微鏡下的形態………………………………28
圖二、單層奈米碳管對人類肺正常細胞之存活率的影響…………………………29
圖三、比較單層奈米碳管以水或十二烷基硫酸鈉當溶劑對人類A549細胞處理48小時後影響存活率…………………………………………………………..30
圖四、比較單層奈米碳管以水或十二烷基硫酸鈉當溶劑對人類A549細胞處理72小時後影響存活率…………………………………………………………..31
圖五、單層奈米碳管誘發A549細胞caspase-3的活化……………………………...32
圖六、單層奈米碳管增加A549細胞的凋亡………………………………………...33
圖七、單層奈米碳管誘發ERK及p38蛋白磷酸化的表現情形……………………34
圖八、單層奈米碳管誘發p53磷酸化蛋白的表達情形…………………………… 35
圖九、單層奈米碳管誘發p53磷酸化蛋白增加,並聚集於細胞核中………………36
圖十、有絲抗原活化蛋白激酶抑制劑對單層奈米碳管所誘發細胞毒性的影響......................................................................................................................37
圖十一、有絲抗原活化蛋白激酶抑制劑對單層奈米碳管造成蛋白活化的影響…38
圖十二、p53抑制劑對單層奈米碳管所誘發細胞毒性的影響……………………39
圖十三、p53抑制劑對單層奈米碳管誘發磷酸p53蛋白活化的影響…………… 40
圖十四、單層奈米碳管誘發Daxx蛋白表達的影響………………………………41
圖十五、單層奈米碳管誘發Daxx蛋白的表達增加,並聚集細胞核及細胞質……42
圖十六、有絲抗原活化蛋白激酶抑制劑對單層奈米碳管誘發Daxx蛋白活化的影響……………………………………………………………………………43
圖十七、p53抑制劑對單層奈米碳管誘發Daxx蛋白活化的影響…………………44
圖十八、單層奈米碳管誘發人類肺細胞凋亡的分子訊息傳遞路徑………………45
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