摘要
癌細胞源自於體內正常細胞開始異常增生並逐步轉變成惡性細胞。這群惡性增生的細胞中，存在極少數量的細胞擁有自我更新與多重分化的特性。除此之外，這一類細胞還具備獨自形成細胞群落發展成腫瘤的潛力以及表現許多與正常幹細胞相同的生物標誌，進而被研究學者定義為癌症幹細胞(Cancer stem cell)。腫瘤癌化與施予化療或手術後癌症的再復發是目前癌症病患死亡的主要原因。另一方面，越來愈多的研究指出癌症幹細胞的新生生能力與抗藥性在癌症治療的抵抗性中扮演重要的角色，因此，癌症幹細胞的研究也越發重要。而如何建立適當的癌症幹細胞辨識與篩選方法並建立合適的寄代培養系統將增進日後生物標記的發現與理解癌症幹細胞自我更新與多重分化的分子調控機制。傳統上共軛焦螢光顯微鏡經常被用於螢光標定影像的判讀，而流式細胞儀則較常使用於分離篩選癌症幹細胞，近期發展之微流體系統更有助於整合辨識、分離篩選與培養於一實驗室晶片提供完整的研究檢測平台。本研究之目的在於設計一癌症幹細胞篩選晶片以成功分離病人腫瘤檢體中的癌症幹細胞，同時以傳統的共軛焦顯微鏡探討癌症幹細胞生物標誌的特徵表現。
論文中，首先使用共軛焦螢光顯微鏡針對三個癌症幹細胞標記，CD133, CD44與Claudin-4在二維與三維細胞培養中的表現量多寡做探討，並選擇表現較高的細胞標記做為下一步在細胞篩選晶片中以免疫磁性分離方法分離癌症幹細胞時所需使用的辨識標記。實驗中使用人類大腸癌細胞HCT-8進行二維單層細胞及三維球體形成 (sphere formation) 培養，運用免疫螢光染色的方式比較生物標記表現。接續，選用表現量最高的標記在微流體晶片系統中進行免疫磁性篩選以分離出腫瘤細胞樣本中的癌症幹細胞。免疫磁性細胞篩選晶片內具有500個由光敏性聚乙二醇二丙烯酸酯經紫外光照射形成的微柱結構，微柱結構中混合了帶有磁性的奈米粒子，能藉由晶片外加磁鐵使微柱結構產生磁性，以微磁柱陣列的方式捕捉被標定磁珠的癌症幹細胞。標定使用的Dynabead磁珠表面上修飾有能夠辨認癌症幹細胞之細胞標記的抗體，磁珠經由抗體連結癌症幹細胞表面抗原使得細胞帶有磁性而能夠被小磁柱吸引捕捉。磁珠搭配微磁柱陣列的設計能將癌症病患血液或腫瘤樣本注入晶片後獲得將近50％的分離效率。希望未來能達成將樣本檢體直接注入微流體晶片後，能篩選分離出癌症幹細胞並直接於晶片中進行培養放大，有助於癌症幹細胞自我更新與多重分化的分子調控機制的研究，以及發展個人化癌症幹細胞藥物抗性篩選平台。

Abstract
Cancer cells initially originate from normal cells that obtain the ability to proliferate aberrantly and become malignant. A subpopulation of these malignant cells has many characteristics of normal stem cells, including self-renewal and differentiation. Also these cells have a potential of growing clonally into tumors and express markers of normal stem cells. Thus, these cells called cancer stem cells (CSCs). Tumor progression and recurrence after surgery or chemotherapy are the main reasons for mortality of cancers. On the other hand, the growing evidence shows that the existence of cancer stem cells is the main cause of cancer resistance to therapy, so it is becoming increasingly important to study CSCs. The ability to identify, isolate, propagate and molecularly characterize CSC subpopulations could improve the discovery of cancer stem cell biomarkers and expand the understanding of the molecular mechanisms that regulate self-renewal and differentiation. Confocal microscopy and microfluidic chip are two novel technologies for imaging and isolation of cells respectively. In this study we used confocal microscopy to investigate the expression pattern of several cancer stem cells markers (CD133, CD44 and Claudin-4) in 2-Dimentional and 3-Dimentional models. The scope of this thesis is to investigate the expression pattern of three cancer stem cell markers using cancer stem confocal microscopy. Then we selected the most expressed marker as a target for immunomagnetic-based cell separation by microfluidic chip technology to isolate CSCs among tumor cells. In this study we used HCT-8 colorectal cancer cell line to generate 2-D monolayer cells, 3-D sphere cells. Our chip possesses 500 micropost structures fabricated by photocrosslinking polymerization of polyethylene glycol diacrylate under the exposure of UV light. These microposts contain iron nanoparticles inside of their structure when a piece of strong magnet placed under the chip, a big magnetic force will be induced in the microposts and give them the ability to capture pre-labeled cells. Tumor cells were treated by antibodies against cancer stem cell markers and then magnetic Dynabeads against these antibodies; consequently the cultured cells labeled with magnetic Dynabeads could be isolated by the chip with approximately 50% of efficiency.

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