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研究生:黃堯章
研究生(外文):Yao-Jhang Huang
論文名稱:利用奈米碳管之光熱特性促進腫瘤細胞衰亡
論文名稱(外文):Carbon Nanotube-Induced Photothermal Therapy for Tumor Cells
指導教授:彭慶安
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:65
中文關鍵詞:腫瘤細胞奈米碳管抗體雷射
外文關鍵詞:Tumor cellscarbon nanotubeantibodylaser
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在眾多的癌症療法中,將奈米粒子輸入癌細胞內,自癌細胞內部進行物理或化學的治療,由於這是在微觀層級以下的療法,因此其效率及潛力具有相當的研究價值。目前癌症治療方式有很多種,最常被醫界認同使用的癌症治療方式可分成四種,即外科手術療法、放射療法、化學療法及免疫(基因)療法,這些療法皆有一定的效用,但也都有一定的限制與瓶頸。因此考慮利用其他的方式(如光、熱等)進行治療為目前癌症治療研究的方向之一。由於奈米碳管具有吸收近紅外光,進而將近紅外光轉為熱能的特性,因此本研究即是將奈米碳管利用腫瘤細胞相應之配體修飾後,與腫瘤細胞共同培養,再以近紅外光照射,利用奈米碳管作為能量轉換的媒介,轉換光能成為熱能,利用過量的熱能使細胞不堪負荷促使細胞的衰亡。
本研究選用神經母細胞瘤細胞stNB-V1,在其表面上有大量的disialoganglioside(GD2)抗原。另外選用無GD2表達之NIH 3T3細胞、BHK-21細胞以及PC-12細胞作為對照組。實驗結果顯示,若碳管上修飾細胞相對應之GD2單株抗體再和上述細胞分別共同培養,在其餘條件皆相同的狀況下,表現有GD2之stNB-V1細胞經由雷射照射後,在雷射照射區域呈現死亡的狀態,而無抗體相對應之對照組細胞則依然存活。由此可見碳管修飾抗體能針對具有標的之腫瘤細胞有顯著的效果,而不會對非標的之細胞造成損害。本研究除了結合奈米碳管、抗體、腫瘤細胞與雷射進行光熱療法實驗外,亦對奈米碳管本身的特性作了部份分析,如碳管酸化前後大小、型態、官能基以及在液體中的分散性之比較。另外也比較了不同濃度的奈米碳管吸收雷射光後對整體溫度的影響,以及觀察碳管在細胞內的狀態。從各種角度反覆驗證與比較此概念之理論與實驗結果之間的異同。
Delivering nanoparticles into cancer cells to proceed physical or chemical therapy is an emerging method in comparison with current cancer therapy methods. In general, strategies used to treat cancer cells can be classified into surgery, radiation, immunotherapy and chemotherapy, which all have their limitation. Therefore, development of innovative treatments for destroying cancer cells remains an important research topic. Because carbon nanotubes (CNTs) can absorb near-infrared light and transform it to heat, we in this study employed CNTs modified with antibody to specifically bind on tumor cells, and then irradiated with 808 nm near-infrared (NIR) laser which can result in malignant cells underwent necrosis due to the existence of carbon nanotubes internalized in cytosol.
Brain tumor stNB-V1 cell line (derived from neuroblastoma) was chosen in this research. On the surface of neuroblastoma cells, there are plenty of disialoganglioside (GD2) glycolipid antigens.. For control groups without GD2 antigen, we selected NIH 3T3, BHK-21 and PC-12 cells. Our results showed that CNTs modified with GD2 monoclonal antibody can be massively internalized into stNB-V1 cells, while this case was not observed for the cells from the control group. After NIR laser irradiation, only stNB-V1 cells in the region exposed to the laser beam were killed. In this research, not only combing carbon nanotube, antibody, tumor cell, and NIR laser to perform thermoablative treatment, but also analyzing the characteristics of carbon nanotubes. Detailed physicochemical analysis of CNTs in size, shape, functionalization, and suspension in different solvents was done prior to the cell experiments. In addition, bulk water temperature profiles in the presence of various CNT concentrations after NIR laser irradiation were measured.
目 錄

口試委員會審定書 ------------------------------------ i

致謝 ----------------------------------------------- ii

中文摘要 ------------------------------------------- iii

英文摘要 ------------------------------------------- v

目錄 ----------------------------------------------- vii

圖目錄 --------------------------------------------- x



第一章 緒論 ------------------------------------------------------------------------------- 1
第二章 文獻回顧 ------------------------------------------------------------------------- 2
2.1. 奈米材料在腫瘤生物治療上的應用 ------------------------------------------ 2
2.1-1. 奈米粒子 --------------------------------------------------------------------- 2
2.1-2. 量子點 ------------------------------------------------------------------------ 3
2.1-3. 奈米馬達與奈米機器人 --------------------------------------------------- 3
2.1-4. 樹形分子 --------------------------------------------------------------------- 4
2.1-5. 幾丁聚醣與乳酸脫氫酶 --------------------------------------------------- 4
2.1-6. 奈米碳管 --------------------------------------------------------------------- 5
2.2. 奈米碳管及其複合材料在生物醫學領域的應用 --------------------------- 6
2.2-1. 奈米碳管的基本結構和特性 --------------------------------------------- 6
2.2-2. 奈米碳管的表面功能化及其對生物分子的作用 --------------------- 6
2.2-3. 奈米碳管複合材料及其生物相容性 ------------------------------------ 7
2.2-4. 奈米碳管支架材料對細胞體外生長的作用 --------------------------- 8
2.2-5. 奈米碳管在原子力顯微鏡探針方面的研究進展 --------------------- 9
2.2-6. 奈米碳管在氣體感測器及生物分子的電化學分析 ----------------- 10
2.2-7. 奈米碳管的生物安全性 -------------------------------------------------- 11
2.3癌症熱療法 ------------------------------------------------------------------------ 12
2.3-1. 熱療法的概念 -------------------------------------------------------------- 12
2.3-2. 磁性微球組織內溫熱療法 ----------------------------------------------- 13
2.3-3. 磁滯效應和鐵磁微球 ----------------------------------------------------- 14
2.3-4. 奈米金粒子於光熱療法中的應用 -------------------------------------- 15
2.3-5. 奈米碳管應用於光熱療法 ----------------------------------------------- 16
2.4. 神經母細胞瘤 ------------------------------------------------------------------- 19
第三章 實驗 ------------------------------------------------------------------------------ 21
3.1. 藥品 ------------------------------------------------------------------------------- 21
3.2. 重要儀器設備 ------------------------------------------------------------------- 22
3.3. 細胞與培養液 ------------------------------------------------------------------- 24
3.4. 實驗步驟 ------------------------------------------------------------------------- 25
3.4-1. 奈米碳管的酸化 ----------------------------------------------------------- 25
3.4-2. 奈米碳管接枝N-羥基琥珀酰亞胺---------------------------------------- 25
3.4-3. 奈米碳管接枝抗體 -------------------------------------------------------- 25
3.4-4. 奈米碳管和細胞共同培養 ----------------------------------------------- 26
3.4-5. 以雷射照射腫瘤細胞 ----------------------------------------------------- 26
3.4-6. 吸收光譜儀測定 ----------------------------------------------------------- 29
3.4-7. 傅立葉轉紅外線光譜之測定 -------------------------------------------- 29
3.4-8. 懸浮性測定 ----------------------------------------------------------------- 30
3.4-9. 熱重法測定 ----------------------------------------------------------------- 30
3.4-10. 粒徑測定 ------------------------------------------------------------------ 30
3.4-11. 界面電位測定 ------------------------------------------------------------ 31
3.4-12. 掃瞄式電子顯微鏡 ------------------------------------------------------ 31
3.4-13. 穿透式電子顯微鏡 ------------------------------------------------------ 32
3.4-14. 升溫曲線測量 ------------------------------------------------------------ 32
3.4-15. 雷射共軛焦顯微鏡分析 ------------------------------------------------ 32
第四章 結果與討論 --------------------------------------------------------------------- 34
4.1. 懸浮性分析 ---------------------------------------------------------------------- 34
4.2. 傅立葉轉紅外線光譜分析 ---------------------------------------------------- 36
4.3. 熱重分析 ------------------------------------------------------------------------- 36
4.4. 吸收光譜分析 ------------------------------------------------------------------- 37
4.5. 電子顯微鏡分析 ---------------------------------------------------------------- 38
4.6. 粒徑分析 ------------------------------------------------------------------------- 40
4.7. 界面電位分析 ------------------------------------------------------------------- 40
4.8. 升溫曲線分析 ------------------------------------------------------------------- 41
4.9. 觀察細胞噬入碳管的狀態 ---------------------------------------------------- 45
4-10. 以鈣黃綠素-AM螢光染劑觀察細胞活性分佈 -------------------------- 47
第五章 結論 ------------------------------------------------------------------------------ 55
第六章 未來展望 ------------------------------------------------------------------------- 57
第七章 參考文獻 ------------------------------------------------------------------------ 58











圖目錄

圖 2-3-1. 奈米碳管應用於光熱療法 ------------------------------------------------ 18
圖 3-2-1. 808 nm雷射裝置示意圖 -------------------------------------------------- 22
圖 3-2-2. 808 nm雷射裝置 ----------------------------------------------------------- 22
圖 3-4-1. 奈米碳管接枝N-羥基琥珀酰亞胺 --------------------------------------- 25
圖 3-4-2. 奈米碳管接枝抗體 --------------------------------------------------------- 26
圖 3-4-3. 細胞利用抗體與受體之間的相互作用噬入奈米碳管 --------------- 27
圖 3-4-4. 細胞噬入碳管後,經由雷射加熱而死亡 -------------------------------- 28
圖 4-1-1. 奈米碳管於各種不同狀況下之懸浮情形 ------------------------------ 35
圖 4-2-1. 碳管酸化前後的傅立葉轉紅外線光譜 --------------------------------- 36
圖 4-3-1. 碳管酸化前後的熱重分析 ------------------------------------------------ 37
圖 4-4-1. 接枝上抗神經節苷脂GD2的碳管以及酸化碳管的吸收光譜 ----- 38
圖 4-5-1. 未酸化與酸化的奈碳在掃描式雷子顯微鏡下之影像 --------------- 39
圖 4-5-2. 未酸化與酸化的奈碳在穿透式電般顯微鏡下之影像 --------------- 39
圖4-6-1. 酸化奈米碳管的粒徑分佈 ------------------------------------------------- 40
圖4-7-1. 酸化奈米碳管於不同酸鹼值下的界面電位---------------------------- 41
圖 4-8-1. 不同濃度奈米碳管以雷射強度6W照射後的升溫曲線 ------------- 43
圖 4-8-2. 不同濃度奈米碳管以雷射強度3W照射後的升溫曲線 ------------- 43
圖 4-8-3. 固定碳管濃度10 μg/ml為以不同功率雷射照射後的升溫曲線 ---- 44
圖 4-8-4. 以雷射強度6W照射不同濃度的碳管溶液之升溫速率曲線圖 ---- 44
圖 4-8-5. 以雷射強度3W照射不同濃度的碳管溶液之升溫速率曲線圖 ---- 45
圖 4-8-6. 固定碳管濃度為10 μg/ml以不同強度的雷射照射碳管溶液之
升溫速率曲線圖 ------------------------------------------------------------ 45

圖 4-9-1. 修飾上抗神經節苷脂GD2的碳管被與PC-12細胞、NIH 3T3
細胞以及BHK-21細胞共同培養後之影像 ---------------------------- 46
圖 4-9-2. 酸化碳管與修飾上抗神經節苷脂GD2的碳管和stNB-V1共同
培養後之影像 --------------------------------------------------------------- 46
圖 4-10-1. 有無對應碳管表面抗體之受體的細胞經雷射照射後呈現不同的
活性分布示意圖 ------------------------------------------------------- 49
圖 4-10-2. 噬入修飾抗神經節苷脂GD2碳管的NIH 3T3細胞經由808 nm
雷射照射後之活性分布 ------------------------------------------------- 50
圖 4-10-3. 噬入修飾抗神經節苷脂GD2碳管的BHK-21細胞經808 nm雷
射照射後之活性分布 ---------------------------------------------------- 51
圖 4-10-4. 噬入修飾抗神經節苷脂GD2碳管的PC-12細胞經808 nm雷射
照射後之活性分布 --------------------------------------------------------- 52
圖 4-10-5. 和酸化碳管共同培養的stNB-V1細胞經808 nm雷射照射後之
活性分布 ------------------------------------------------------------------- 53
圖 4-10-6. 噬入修飾抗神經節苷脂GD2碳管的stNB-V1細胞經808 nm雷
射照射後之活性分布 ---------------------------------------------------- 54
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