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研究生:陳雅筠
研究生(外文):Ya-Yun Chen
論文名稱:於HepG2肝癌細胞株評估調控式電熱治療儀(mEHT)與不同形狀的金奈米粒子之合併效應
論文名稱(外文):Evaluate the combination effect of modulated electro-hyperthermia (mEHT) and gold nanoparticles with different shapes in HepG2 hepatoma cell line
指導教授:陳傳霖陳傳霖引用關係
指導教授(外文):Chuan-Lin Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學影像暨放射科學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:55
中文關鍵詞:金奈米粒子調控式電熱治療儀細胞毒性細胞凋亡
外文關鍵詞:gold nanoparticlesmodulated electro-hyperthermiacytotoxicityApoptosis
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中文摘要
目的:近年來熱治療(hyperthermia)已實際運用於臨床癌症輔助治療上,而調控式電熱治療儀(modulated electro-hyperthermia, mEHT)為其中一種熱治療設備,其特性是可專一於腫瘤病灶區升溫達到熱輔助治療成效。目前金奈米粒子(gold nanoparticle, AuNP)已被廣泛作為奈米藥物載體,雖然過去研究報導曾運用金奈米粒子於電磁射頻場(radiofrequency, RF)中並具有顯著升溫現象,但尚未有金奈米粒子合併mEHT之研究,因此本研究欲合成不同形狀之金奈米粒子(球狀、刺蝟狀及棒狀),同時結合mEHT探討其對腫瘤細胞之影響。
方法:利用不同方式製成球狀(SAuNP)及刺蝟狀(UAuNP)之金奈米粒子,可得到較為均一金奈米粒子,另外棒狀金奈米粒子則可由Sigma-Aldrich購買獲得,並利用紫外光可見光光光譜(UV-visible)、動態光散射儀(DLS)、電子顯微鏡(TEM)及電漿質譜(ICP-MS)儀進行金奈米粒子特性分析。再以Oncotherm EHY-100此一mEHT設備對不同濃度及形狀之金奈米粒子探討其升溫效應,同時於HepG2腫瘤細胞探討結合金奈米粒子對細胞存活與凋亡之影響。分析方式以MTT決定存活率及 Annexin V-FITC染色後以流式細胞儀計讀凋亡比例。
結果:三種金奈米粒子合成法Turkevich、Seed growth與對二苯酚還原皆能合成出不同粒徑之金奈米粒子,經比較後,以對二苯酚還原合成法較能控制粒徑大小,並將條件最佳化,以特定奈米金晶種數量,254 μmol四氯酸金,22 nmol檸檬酸鈉及特定濃度對二苯酚3 μmol與30 μmol,可分別獲得粒徑約為50 nm之球狀及刺蝟狀金奈米粒子。將不同形狀粒徑約為50 nm之奈米粒子於mEHT中進行升溫測試,升溫速率皆約0.4˚C/min並無顯著差異。以不同濃度由1.56至100 ppm之50 nm不同形狀之金奈米粒子溶液與肝癌細胞(HepG2)共同培養24小時後亦無明顯細胞毒性產生。配合不同加熱方式、水浴(37˚C,42˚C)及調控式電熱療(mEHT)與不同形狀濃度為25 ppm金奈米粒子(球狀、刺蝟狀、棒狀)共同培養HepG2肝癌細胞後,比較腫瘤細胞之變化,相較於水浴加熱,在有無奈米金存在下,經mEHT處理的組別腫瘤細胞皆有顯著性傷害 (存活率下降及凋亡比例增加),但不同形狀之間對於細胞存活與凋亡比例則無顯著性差異;若以金奈米粒子與肝癌細胞預先培養24小時後,再以不同加熱方式處理,在水浴37˚C及42˚C結果與上述金奈米粒子共同培養組別相似,而在mEHT下則可觀察到經奈米粒子預先培養之細胞其細胞存活率明顯較高且細胞凋亡比例下降,其中以早期凋亡比例有顯著降低,顯示預先培養之金奈米粒子進入細胞後能減少mEHT對細胞之傷害。
結論:本研究成功利用對二苯酚還原合成法合成粒徑大小為50 nm球狀與刺蝟狀金奈米粒子。合併mEHT升溫試驗結果顯示不同形狀金奈米粒子(球狀、刺蝟狀、棒狀)及濃度皆不影響mEHT升溫速率。金奈米粒子合併mEHT之細胞傷害結果顯示,共同培養裡有無金奈米粒子對HepG2細胞皆有明顯傷害但無顯著差異,而預先培養金奈米粒子於HepG2細胞中則將大幅降低mEHT對細胞之傷害效應。
Abstract
Purpose: Hyperthermia (HT) has been used as an adjuvant cancer therapy in the clinic. The Modulated electro-hyperthermia (mEHT) is a new loco-regional electromagnetic hyperthermia method for selectively targeting cancer cell hyperthermia treatment. Gold nanoparticles (AuNP) have been demonstrated as a promising nano-drug carrier and several studies indicated that AuNP can promote heat generation as exposed to the radiofrequency with the better result of cell killing effect. Till now, the AuNP was combined with mEHT for cancer cell hyperthermia therapy hasn’t been investigated and might possess synergistic effect for mEHT treatment. In this study, the different shapes (spherical, urchin-like or rod) AuNP were prepared and treated with HepG2 cell line under various HT conditions (including water bath and mEHT) to investigate the differences in the biological effect.
Methods: The homemade spherical and urchin-like AuNPs were prepared according to different methods. The homogenous sizes and sharps AuNPs were obtained from the seed-mediated growth method with hydroquinone. Gold nanorod was purchased from Sigma-Aldrich. All nanoparticles were characterized by UV-VIS absorption spectrometry, dynamic light scattering (DLS), transmission electron microscope (TEM) and inductively coupled plasma mass spectrometry (ICP-MS). Oncothermia EHY-100 was used for investigating the heating effect of AuNP with different shapes and the killing effect of HepG2 hepatoma cells after the combination AuNP with mEHT. The cell survival was analyzed by MTT assay and Annexin V staining.
Results: Gold nanoparticles were synthesized by Turkevich, seed-growth and hydroquinone reduction method. Among these methods, the seed-mediated growth approach with hydroquinone reduction method had the advantage in controlling particle sizes and sharps. The spherical and urchin-like gold nanoparticles were synthesized by adjusting the amount of hydroquinone. The diameter of ~50 nm spherical and urchin-like gold nanoparticles were obtained from 5x10^11~1x10^12 gold seeds reacted with 254 μmol HAuCl4, 22 nmol sodium citrate and 3 μmol or 30 μmol hydroquinone in 10 ml deionized water, respectively. The heating rate of AuNPs was around 0.4˚C/min by mEHT treatment and no significant differences were observed between deionized water and various sharps AuNPs solutions. After 24 h incubated with 50 nm AuNPs solution, the concentration of 1.56 to 100 ppm showed negligible cytotoxicity to HepG2 cells and no difference was obvious among various shapes of AuNP. HepG2 cells were exposed to HT treatment (water bath and mEHT). Compared with water bath (42˚C) treatment, the mEHT treatment showed significantly reduced survival ratio. While no significant synergistic effect on cell killing of HepG2 cell was noticed in the co-incubation with various AuNPs as mEHT treatment. However, compared to the control group (without AuNP incubation) or the co-incubation groups, the cell-killing effect of mEHT was significantly decreased in those groups which HepG2 cells pre-incubated with AuNPs for 24 h and then exposed to mEHT treatment. The results of annexin-V flow cytometry revealed that percentage of early apoptotic cells significantly reduced.
Conclusion: This study has demonstrated that 50 nm of spherical and urchin-like gold nanoparticles could be synthesized by adjusting the reaction concentration of hydroquinone in the hydroquinone reduction method. Compare to water, there is no significant heat generation in different shapes of AuNPs after exposing to mEHT. Similar cell damage effects of mEHT in HepG2 cells were noticed between the control group (without AuNPs) and the co-incubation groups, while high cell survival and low apoptosis ratio was observed in the pre-incubation groups.
目錄
目錄 i
圖目錄 iv
表目錄 vii
中文摘要 viii
Abstract x
第 1 章 緒論 1
1.1. 金奈米粒子簡介與應用 1
1.2. 癌症的輔助治療-熱治療 (hyperthermia) 3
1.3. 研究目的 9
第 2 章 材料與方法 10
2.1. 實驗材料 10
2.2. 金奈米粒子之製備 12
2.3. 金奈米粒子物理性質分析 17
2.4. 金奈米粒子穩定度檢測 18
2.5. 調控式電熱療儀(mEHT)之設置 19
2.6. 不同形狀之金奈米粒子於 mEHT 之升溫測試 19
2.7. 金奈米粒子合併 mEHT 加熱之細胞實驗 20
2.8. 細胞粒線體活性分析(MTT assay) 21
2.9. 細胞凋亡分析(Cell Apoptosis) 22
第 3 章 結果 23
3.1. 金奈米粒子製備 23
3.2. 金奈米粒子之穩定度測試 32
3.3. 金奈米粒子濃度定量 32
3.4. 金奈米粒子於 mEHT 之升溫測試 33
3.5. 金奈米粒子之細胞生物相容性 34
3.6. HepG2 肝腫瘤細胞經金奈米粒子處理合併 mEHT 加熱對細
胞存活之影響 35
3.7. 腫瘤細胞(HepG2)經金奈米粒子處理合併 mEHT 加熱之機制
探討 37
第 4 章 討論 41
4.1. 金奈米粒子之製備 41
4.2. 不同形狀之金奈米粒子在 mEHT 的升溫效應 43
4.3. HepG2 經不同形狀之金奈米粒子合併 mEHT 加熱之生物效
應 46
第 5 章 結論 49
第 6 章 參考文獻 50
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