請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62667
標題: | 螢光奈米鑽石的基因毒性測試及其在乳癌癌症幹細胞辨識、純化與追蹤方面的應用 The Genotoxicity of Fluorescent Nanodiamond and its Use in the Identification, Isolation, and Tracking of Breast Cancer Stem Cells. |
作者: | Hsiao-Wen Lee 李筱雯 |
指導教授: | 張煥正(Huan-Cheng Chang) |
關鍵字: | 螢光奈米鑽石,乳腺癌幹細胞,細胞追蹤,基因毒性, Fluorescent nanodiamond,Cancer stem cell,Genotoxic,Cell tracking, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 螢光奈米鑽石(fluorescent nanodiamond)是一種新穎的奈米碳材,利用高能量的氦離子束及高溫鍛燒使100nm Type Ib的鑽石產生高濃度的帶負電的氦-空缺(NV-)缺陷中心,缺陷的特性使粒子在經過黃綠光的激發下會放出遠紅外光的特性,此種光學性質沒有光漂白(no photobleaching)、沒有光閃爍(no photoblinking);除了獨特的光學特性外,奈米螢光鑽石為碳系的相關材料,表面容易進行化學修飾,並且與生物相容性高,因此近年來廣泛的使用在標記細胞與長時間的追蹤。
本篇論文第一部分中,主要探討奈米螢光鑽石在標記細胞後是否會影響、破壞細胞的脫氧核醣核酸(DNA, deoxyribonucleic acid),所做的基因毒性測試。在標記奈米螢光鑽石於人類成纖維細胞(HFW, Human Fibroblast)後,觀察細胞的生長速度(proliferation)、存活率(survival)是否有受影響,除此之外,利用彗星法(comet assay)、微核細胞法(micronucleus test)來判斷是否脫氧核醣核酸(DNA, deoxyribonucleic acid) 會受到奈米螢光鑽石標記的影響。由結果顯示,奈米螢光鑽石並不會影響細胞生長及產生毒性。 第二部分中,則是利用奈米螢光鑽石特有的螢光性質、克服一般螢光染料會隨著時間發生光漂白、光閃爍,所以不利於長時間的追蹤的缺點,且與奈米螢光鑽石高生物相容性等多項優點結合,設計一個新穎的策略來追蹤、純化乳癌幹細胞。乳癌幹細胞具有休眠、分化的特性,存在於癌症腫瘤中,被認為是誘發腫瘤生長的主因之一,因此純化乳癌幹細胞為癌症治療的重要步驟。本論文中結合螢光奈米鑽石和乳腺癌幹細胞,在不影響乳癌幹細胞情況下利用奈米鑽石的螢光性來判斷細胞的分化時間、ALDH activity的表現量、和觀察克隆(clonogenicity)的成長。並且和一般用來追蹤細胞的螢光染料CFSE(carboxyfluorescent succinimidy ester)做比較。結果顯示螢光奈米鑽石不會改變乳癌癌症幹細胞的生長狀態,然而CFSE不只會影響細胞的生長情況,還會造成部分細胞死亡凋零。此外,利用EDU (5-ethynyl-2-deoxyuridine)染DNA的策略來追蹤細胞分裂狀況以便判斷細胞是否有休眠狀態,在24天的追蹤後,仍有一群細胞且具有高劑量的螢光奈米鑽石且其DNA並未進行分裂,這表示奈米螢光鑽石可以在不影響生物特性下追蹤一群具有休眠狀態的特殊細胞,而後可以藉著流式細胞儀將仍有活力的細胞分離出來做後續研究使用。此奈米螢光鑽石策略可以提供生物學家更有效率純化乳癌癌症幹細胞,以利乳癌治療方法研發能有更快速的進展。 Fluorescent nanodiamond (FND) has recently emerged as a novel fluorescent probe for biological applications. In this work, high energy helium ion beam was used to irradiate 100-nm type Ib nanodiamonds, followed by annealing, to create a high density of negatively charged nitrogen-vacancy (NV−) defect centers as fluorophores. The center emits far infrared fluorescence under excitation by green yellow light with excellent optical properties such as neither photobleaching nor photoblinking. Moreover, the surface of FND is easy to be functionalized for specific cell targeting. These characteristics, along with the nontoxicity, make FND distinct from conventional fluorescent probes in cell labeling and long-term tracking applications. This thesis is composed of two parts. In the first part, a series of genotoxicity experiments (including micronucleus and comet assays) have been carried out to understand if FND would cause any damage to the deoxyribonucleic acid (DNA) of FND-labeled cells. Human fibroblasts were chosen to study the effects in vitro. The results, together with the measurement for proliferation and survival rates, indicate that FNDs are not genotoxic and do not impart the biological function of the cells. The second part involves FND labeling of quiescent cancer stem cells (CSCs), which have been considered to be one of the sources of tumor formation. However, the isolation tactics of CSCs are still incomplete owing to the fact that the fluorescence intensity of commonly used biomarkers would decay with time. Moreover, some molecule-based biomarkers would affect the basic properties of the cells. In light of the need for a photostable and chemically stable biolabel, we have developed and used FNDs for the identification, isolation, and characterization of quiescent breast tumor CSCs. By using the new FND-labeling method, we have discovered a special group of cells which contain a relatively high dosage FNDs and undivided DNA after 24 days in vitro observation. The result demonstrates that FNDs can be utilized as an effective nanoprobe to purify and isolate quiescent CSCs for future cancer therapy research. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62667 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-102-1.pdf 目前未授權公開取用 | 2.48 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。