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系統識別號 U0026-2408201914302300
論文名稱(中文) 奈米粒子用於體外標記神經膠質母細胞瘤的潛在應用
論文名稱(英文) The potential application of using nanoparticle to label glioblastoma in vitro
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 107
學期 2
出版年 108
研究生(中文) 吳宜璇
研究生(英文) Yi-Syuan Wu
電子信箱 polyingreen@gmail.com
學號 T96031046
學位類別 碩士
語文別 英文
論文頁數 77頁
口試委員 指導教授-司君一
口試委員-陳宣燁
口試委員-許鍾瑜
口試委員-鄧志娟
中文關鍵字 神經膠質母細胞瘤  表皮成長因子接受器  功能性奈米粒子  拉曼散射  表面增強拉曼散射  雷射  光熱治療 
英文關鍵字 Glioblastoma  Epithermal Growth Factor Receptor (EGFR)  Functionalized gold nanoparticles  Raman scattering  Surface-enhanced Raman scattering  Laser  Photothermal therapy 
學科別分類
中文摘要 星狀膠細胞(Astrocyte)為神經膠細胞的其中一種,因形狀似星型而得其名,主要 功能為供給神經細胞養分、與外被細胞(Pericyte)、血管內皮細胞一同構成血腦障壁 (Blood-Brain-Barrier)的結構,保護中樞神經系統。而多型性神經膠質母細胞瘤 (Glioblastoma, GBM)便是因星狀膠細胞分裂時產生異常所產生的疾病,病理學上可見 壞死組織及大量增生的血管,且因其特殊的浸潤性生長侵略正常腦組織,導致完整清 除腫瘤的難度非常高,病患存活率偏低。
從腫瘤內有大量血管此一特性,加上腫瘤旁血管的血腦障壁有崩解的情形產生, 而我們也可以從文獻中知道 GBM 細胞膜上會表現大量的表皮細胞生長因子接受器 (Epidermal growth factor receptor, EGFR)。因此利用這種特性,在本實驗中,我們使用 外圍接上 anti-EGFR 抗體的功能性奈米金粒子標記腫瘤細胞,並使用光熱療法 (Photothermal therapy)殺死腫瘤細胞,達到治療的療效。
在實驗一開始,首先先以細胞染色法及西方墨點法確認大鼠 GBM 細胞(CNS-1) 是否有大量表現 EGFR,再者,我們必須確認之後要使用的功能性奈米金粒子是否具 有特異性,能接上 GBM 細胞膜表面的 EGFR,因此使用修飾過後的酵素連結免疫吸 附分析法、布萊德福蛋白質定量法以及透過掃描式電子顯微鏡檢測,但這些方式無法 精確的確認功能性奈米粒子的特異性,因為第一代核心-衛星金粒子叢集(core-satellite assemblies, CSAG1)可能因結構不穩定,導致聚集及沈降的機率增高,影響實驗結果, 因此第二代 CSA 被研發後,穩定性提升,因此在往後的實驗中會使用 CSAG2 來檢測 特異性及應用在光熱治療。在更進一步確認特異性的實驗中,我們使用 CSAG2@anti-EGFR 以及控制組 CSAG2@IgG 兩種奈米粒子來檢測。實驗結果表示最適合檢測的參數為:當 1.8×109 個 CSAG2@anti-EGFR 與細胞反應 3 小時,再以照度 410 W/cm2 的 660nm 雷射照射,每一點照射時間為 1 分鐘,共照射 7 個點,或每一點照 射時間為 3 分鐘,共照射 5 個點的效果最好。
再確認 CSAG2@anti-EGFR 的特異性後,我們使用共培養系統將 CNS-1 及大鼠星 狀膠細胞種在同一個 96well 的培養孔,藉此模擬正常細胞與癌細胞的交界處。實驗 結果顯示 CSAG2@anti-EGFR 增加至 2.5×109 個,與細胞培養 3 小時,再照射 410 W/cm2 的 660nm 雷射能提升腫瘤細胞的死亡率至約 53%左右,表示 CSAG2@anti-EGFR 具有 運用在標記腫瘤細胞的潛力。
由於人類肉眼很難分辨正常細胞及腫瘤細胞,因此醫師及科學家們會依賴某些工 具去運用在生物醫學領域中。拉曼散射是一種非彈性散射,因其訊號微弱,所以被發 現之後沒有被廣泛運用,直至表面增強拉曼散射(SERS)被發現。奈米金粒子是一種被 常運用在 SERS 中的物質,藉由表面電漿共振達到訊號放大的效果。SERS 最大的特 點為在水中訊號不被干擾,因此或許在未來可以運用在人體中,而運用此特點, Cy5-CSAG3@anti-EGFR 被研發,藉由 Cy5 可放大拉曼訊號的特徵, Cy5-CSAG3@anti-EGFR 可以被運用於標記追蹤腫瘤細胞或轉移至其他部位的癌細胞。 而在本研究中,我們使用共培養系統確認拉曼影像引導的光熱治療效果,並證明 Cy5-CSAG3@anti-EGFR 未來被應用在臨床上的潛力。
英文摘要 Astrocyte is one kind of star-shaped neuroglial cell in the brain. Their main functions are maintaining the healthy central nervous system (CNS)[1], forming the structure blood-brain-barrier with pericytes and vessel endothelial cells to protect the brain. Glioblastoma (GBM) is a disease due to abnormal cell division of astrocytes. There are necrotic tissue and massive vascular proliferation inside the tumor. Because the infiltrating property, removal of the whole cancer cells is difficult in surgery. The patients’ survival rate is low. From the literature search, the epidermal growth factor receptor (EGFR) is amplified at the cell membrane of GBM, and the blood-brain-barrier (BBB) next to tumor has been collapse by the pressure from tumor. In this study, I used these properties including massive vascular proliferation in the tumor, and used the functional gold nanoparticles (Core-satellite assemblies, CSA) conjugated with anti-EGFR antibody to target tumor cells. In the beginning of the experiment, I used immunocytochemistry and Western blot to confirm rat GBM cells (CNS-1) that expressed EGFR. Next, I tried to confirm the specificity of CSAG1@SiO2@anti-EGFR by modified ELISA, Bradford assay and SEM, but the results couldn’t totally confirm the specificity of CSAG1@SiO2@anti-EGFR due to the unstable structure of CSAG1. Therefore, CSAG2 were developed. To further confirm the specificity, the CSAG2@anti-EGFR and CSAG2@IgG were used to test. The results showed that the suitable condition was after reacted with 1.8×109 CSAG2@anti-EGFR for 3hours, then 410W/cm2 of 660nm laser irradiated for 3minutes, 5points or 1minute, 7points. After confirming the specificity of CSAG2@anti-EGFR, I used a co-culture system that seeded CNS-1 and rat astrocytes in the same well to imitate tumor margin in vivo. The results showed that cells reacting with 2.5×109 CSAG2@anti-EGFR for 3hours and irradiated 410W/cm2 of 660nm laser for 3minutes 5points and 1minute 7points would increase the cell death rate, suggesting that the CSAG2@anti-EGFR have potential in targeting tumor cells. To distinguish tumor cells from normal cells by human eyes is difficult. Therefore the doctors or scientists developed tools. Raman scattering is a phenomenon of inelastic scattering. Due to the weakness of signal, this technique wasn’t used widely in the past few decades, until surface-enhanced Raman scattering (SERS) was discovered. Gold nanoparticle is one of the materials that have been widely used in SERS via the surface plasma resonance. The main advantage of SERS is stable signal in water. Human body contains 70% of water, suggesting SERS signal might be applied to human. The Cy5-CSAG3@anti-EGFR was developed to enhance Raman signal and could be applied not only to track GBM cells but also other cancer cells with metastasis. In this study, I used the co-culture system to confirm the effect of Raman guided photothermal therapy, and to test the potential of Cy5-CSAG3@anti-EGFR in future clinical application.
論文目次 Abstract in Chinese...............................................................................III
Abstract in English................................................................................V Acknowledgment...................................................................................VII
Table of Contents................................................................................VIII
List of Figures.....................................................................................XII Introduction
Glioblastoma Multiforme (GBM) ............................................................1
Receptor Tyrosine Kinases (RTKs) ..........................................................1
Raman Scattering..................................................................................2
Surface-enhanced Raman Spectroscopy (SERS)..............................................3 Raman Tags......................................................................................3
Cancer Nanomedicine...............................................................................4 Photothermal therapy (PTT).....................................................................5
Hypothesis and Specific Aims...........................................................................6 Experimental Design.....................................................................................7 Materials and Methods
Cell Cultures.....................................................................................9
Co-culture of CNS-1 and Rat astrocyte.....................................................10 Immunocytochemistry..........................................................................10
Western Blot...................................................................................11
Structural Synthesis of CSAG1@SiO2@antibody.........................................11 Structural Synthesis of CSAG2...............................................................13 Structural Synthesis of Cy5-CSAG1.........................................................13 Structural Synthesis of Cy5-CSAG2.........................................................14 Structural Synthesis of Cy5-CSAG3.........................................................15 Modified Direct ELISA......................................................................15
Bradford assay.................................................................................16
Trypan Blue Exclusion Assay...............................................................16 Transmission Electron Microscopy (TEM) ...............................................16 Scanning electron microscopy (SEM) .....................................................17 Photothermal therapy.........................................................................18 Photothermal therapy of co-culture system................................................18 Raman guided photothermal therapy.......................................................19 Statistical analysis.............................................................................19
Results
The different expression of the Receptor Tyrosine Kinases (RTKs) of GBM and Human Astrocyte..............................................................................25
The synthesis of functional gold nanoparticles............................................26
After functionalized, the GNPs showed the selectively in targeting cancer cells by modified ELISA...............................................................................27
After functionalized, the GNPs showed the selectively in targeting cancer cells by Bradford assay................................................................................28
After functionalized, the GNPs showed the selectively in targeting fixed cancer cells by SEM .................................................................................29
After functionalized, the GNPs showed the selectively in targeting living cancer cells by SEM...................................................................................30
The morphology changed after cells reacted with GNPs................................30
The photothermal efficacy of CSAG1@COO- in CNS-1.................................31
The specificity of CSAG1@Ab via PTT test..............................................31
The specificity of CSAG2@Ab via PTT test..............................................32
The specificity of CSAG2@Ab in CNS-1 and Rat astrocyte by PTT..................33 The specificity of CSAG2@anti-EGFR after raised the concentration and
decreased the reaction time by PTT........................................................34
PTT test with CSAG2@anti-EGFR in co-culture system to imitate tumor margin.........................................................................................34
PTT test with Cy5-CSAG2@anti-EGFR in co-culture system to imitate tumor margin.........................................................................................35
PTT test of Cy5-CSAG2@anti-EGFR and the specificity test of Raman shift after increased the reaction time with cells in co-culture system.............................35
Raman effect guided PTT via Cy5-CSAG3@anti-EGFR in co-culture system.......36
Figures.............................................................................................38
Discussion.......................................................................................67 Conclusion.......................................................................................71 References.......................................................................................72
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