臺灣博碩士論文加值系統

膠質母細胞瘤 ( Glioblastoma multiforme ) 是原發性腦瘤中最常見，也是最為惡性的腦瘤；而腫瘤中的癌症幹細胞被認為是造成癌症復發的原因，因此希望能透過放射線治療及放射線增效劑的結合有效抑制經手術後殘留的癌幹細胞的生長。
我們發現常用化療藥物帝盟多 (TMZ) 對於本實驗中所選用之膠質母細胞瘤之癌幹細胞 (GSC) 有較低的細胞毒性(IC50 = 870 μM)，然而正常星狀膠細胞 (SVG p12) 對於TMZ敏感性相對較強 (IC50 = 380 μM)，因此使用TMZ化療藥物針對GSC進行治療會有較差的療效。所以本實驗欲透過奈米金-二氧化矽複合載體(Au@mSiO2)SiO2作為輻射增效劑，結合放射線治療增加對於GSC之細胞毒性。在研究中可發現(Au@mSiO2)SiO2 在GSC細胞與SVGp12細胞間即有良好的毒殺選擇性，然而對於結合輻射治療並沒有太大的幫助，但對於正常細胞亦不造成影響。在細胞週期的測定中可發現GSC細胞施予載體並結合輻射治療後，細胞週期G2/M累積比例稍有增加，推測載體仍然對於輻射增效有幫助，但可能由於後期的DNA修復導致效果不顯著，這個部分仍須再進行研究。

Glioblastoma multiforme is the most common and most malignant primary brain tumors. Cancer stem cells in the tumor are thought to be the cause of cancer recurrence. We hope to combine the radiation enhancers with radiation therapy to inhibit the growth of cancer stem cells, which were remained after surgery.
We found that the commonly used chemotherapeutic drugs Temozolomide (TMZ) had lower cytotoxicity (IC50 = 870 μM) for the glioblastoma cancer stem cells (GSC), whereas the astrocytes (SVG p12 ) is more sensitive to TMZ (IC50 = 380 μM), so the use of TMZ for GSC treatment will have a poor effect.
The experiment aims to increase the cytotoxicity to GSC by using Au-silica composite nanoparticles (Au@mSiO2) SiO2 as radiation enhancers, combined with radiation therapy. In the study, we found that there was a good selectivity between GSC cells and SVGp12 cells throughout the usage of (Au@mSiO2) SiO2, but it did not help much for radiation enhance, although it did not affect normal cells.
In the determination of cell cycle, the G2 / M arrested of GSC cells increased slightly during the combination therapy, suggesting that the vector is still helpful for radiation enhancement, but DNA may be repaired due to the GSC characteristic, and this part still needs to be studied.

目錄
致謝.....I
摘要.....II
Abstract.....III
目錄.....IV
圖目錄.....VII
表目錄.....IX
第一章 緒論.....1
第二章 文獻回顧.....2
2.1 膠質母細胞瘤.....2
2.2 癌症幹細胞.....4
癌症幹細胞的輻射抗性.....7
癌症幹細胞的化學抗性.....8
2.3 輻射.....9
X-射線.....10
放射線治療.....11
2.4 放射線增效作用.....13
金奈米顆粒的輻射增效.....14
二氧化矽的輻射增效.....16
第三章 材料與方法.....17
3.1 實驗設計.....17
3.2 材料.....18
3.3 儀器.....19
3.4 實驗方法.....20
載體合成.....20
奈米金-多孔性二氧化矽 ( Au@mSiO 2 ) 合成.....20
異硫氰酸熒光素修飾(Au@mSiO 2- FITC).....21
無孔洞性二氧化矽殼層修飾( (Au@mSiO 2 ) SiO 2 ).....21
載體分析.....23
動 態 光 散 射 粒 徑 分 析 儀 ( Dynamic Light Scatter Analyzer，DLS ).....23
界面電位分析儀 ( Zeta Potential Analyzer).....23
穿 透 式 電 子 顯 微 鏡 / 能 量 散 射 光 譜 儀 (Transmission electron microscope，TEM / Energy Dispersive Spectrometer，EDS).....23
傅 立 葉 轉 換 紅 外 光 譜 儀 ( Fourier transform infrared spectroscopy，FT-IR ).....24
X 光繞射儀 ( X-ray diffractometer，XRD ).....24
體外細胞實驗.....25
細胞培養.....25
帝盟多化療藥物毒性測試.....25
(Au@mSiO 2 ) SiO 2 載體細胞毒性測試.....26
共軛焦顯微鏡觀察細胞載體吞噬情形.....26
X 光輻射治療測試.....27
細胞週期測定.....28
DNA fragmentation assay.....28
第四章 結果與討論.....29
4.1 (Au@mSiO 2 ) SiO 2 載體材料結構分析.....29
4.2 (Au@mSiO 2 ) SiO 2 載體材料性質分析.....32
FT-IR 材料分析.....32
EDS 元素分析.....34
XRD 材料分析.....35
4.3 體外細胞實驗.....36
帝盟多化療藥物毒性測試.....36
(Au@mSiO2) SiO2 載體背景毒性測試.....40
共軛焦顯微鏡觀察細胞載體吞噬情形.....42
X 光輻射治療測試.....44
細胞週期測定.....46
(Au@mSiO 2 ) SiO 2 及 XR 治療造成之細胞凋亡情形.....51
第五章 結論.....54
參考資料.....55
附錄.....60


圖目錄
Fig. 2-1 Distribution of all primary CNS gliomas. [1].....2
Fig. 2-2 A computer-aided volumetric approach to measure an irregularly shaped glioblastoma. [8].....3
Fig. 2-3 Origin, self-renewal, and dedifferentiation of GSCs. [4].....5
Fig. 2-4 Cancer stem cells cause tumour regrowth after apparently successful therapy. [13].....5
Fig. 2-5 (A) The activation state of the checkpoint response in matched CD133 - and CD133 + cells from glioblastoma specimens was assessed before treatment ( - ) and 1 h after 3 Gy of IR ( + ). (B) The presence of DNA damage of CD133 - and CD133 + cells at sequential time points after irradiated with IR was assessed by alkaline comet assay. (C) Quantification of the percentages of cells with comet tails at different time points after IR in CD133 - and CD133 + populations. Data are the means ± s.d. (n=100 cells in three trials; *P < 0.001; **P < 0.002). [5].....7
Fig. 2-6 (A) ABCG2 expression in neurospheres. (B) Evaluation of intracellular Photofrin concentration. (*p < 0.01 versus the control and **p < 0.05 versus the FTC-group). [16].....8
Fig. 2-7 Radiation in daily life......9
Fig. 2-8 X-ray generator......10
Fig. 2-9 X-ray causes DNA damage. [17].....11
Fig. 2-10 Single-cell survival data for various stages of the cell cycle.[18].....12
Fig. 2-11 Interaction of X-rays with high-Z material nanoparticles. [25].....13
Fig. 2-12 ROS production in aqueous suspensions: use of PhOH, FFA, and His as scavengers. Relative scavenger concentration versus X-ray irradiation dose for solutions of 10 -4 M PhOH (filled circle), 10 -4 M His (filled square), 10 -4 M FFA (filled triangle), and 0.50 g/L MMASi-NP suspensions containing 10 -4 M PhOH (open circle), 10 -4 M His (open square), and 10 -4 M FFA (open triangle).[27].....16
Fig. 3-1 Experimental Design......17
Fig. 3-2 Flow chart of (Au@mSiO 2 ) SiO 2 synthesis。.....22
Fig. 4-1 TEM image of (A) Au@mSiO 2 and (B) (Au@mSiO 2 ) SiO 2 , (C) HR-TEM image of (Au@mSiO 2 ) SiO 2 , (D) size distribution, (E) zeta potential analysis. 30
Fig. 4-2 FTIR spectra of the Au@mSiO 2 with CTAB, Au@mSiO 2 remove CTAB, Au@mSiO 2 -FITC, (Au@mSiO 2 -FITC) SiO 2.....33
Fig. 4-3 STEM-EDS line mapping of (Au@mSiO 2 ) SiO 2......34
Fig. 4-4 XRD pattern of (Au@mSiO 2 ) SiO 2.....35
Fig. 4-5 (A) Cytotoxicity of TMZ against GSC and Astrocyte at different concentrations. (B) Determination of IC 50 value through the cell viability assay of TMZ against GSC and Astrocyte......39
Fig. 4-6 Cytotoxicity of (Au@mSiO 2 ) SiO 2 against GSC and Astrocyte at different concentrations, the cell viability was detected by 3 days after treatment......41
Fig. 4-7 (Au@mSiO 2 ) SiO 2 localization detection. Maximum intensity projection images of (A)GSC (C) SVG p12, and section images of (B) GSC (D) SVG p12 treated with 25 μg/mL (Au@mSiO 2 -FITC) SiO 2 for 6 hours, then stain with phalloidin-TRITC and Hoechst 33258......43
Fig. 4-8 Cytotoxicity of (Au@mSiO 2 ) SiO 2 and radiation therapy against GSC and Astrocyte. Cells were treated with 25 μg/mL (Au@mSiO 2 -FITC) SiO 2 for 6 hours, then followed by 4 Gy radiation therapy (XR), the cell viability was detected by 3 days after XR therapy......45
Fig. 4-9 Irradiation induces G1 and G2 cell cycle checkpoint activation and DNA repair. [39].....46
Fig. 4-10 Cell cycle of different hours after GSC treated with 4 Gy XR therapy and (Au@mSiO 2 )SiO 2 + 4 Gy XR therapy......48
Fig. 4-11 Cell cycle of different hours after astrocyte treated with 4 Gy XR therapy and (Au@mSiO 2 )SiO 2 + 4 Gy XR therapy......49
Fig. 4-12 Cell cycle phase percentage of (A) GSC, (B) SVG p12 cell.....50
Fig. 4-13 Schematic of the DNA staining procedure using the TUNEL assay. [40].....51
Fig. 4-14 DNA fragmentation of SVG p12 cells and GSC were analyzed by TUNEL analyses (green). Nuclei of cells were stained with DAPI (blue). Scale bar: 50 µm......53


表目錄
Table 2-1 Mechanisms of Resistance in GSCs. [4].....6
Table 2-2 High-Z nanoparticless and their in vitro radiation sensitizing effects. [21].....15
Table 4-1 Size distribution and zeta potential analysis.....31

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