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研究生:游嘉豪
研究生(外文):Chia-Hao Yao
論文名稱:微結構孔盤應用於體外檢測腫瘤細胞之爬行能力
論文名稱(外文):An In-vitro Functional Assay to Assess Migration of Cancer Cells using the Micro Gap Plate
指導教授:胡文聰胡文聰引用關係
口試委員:李雨許聿翔
口試日期:2016-07-20
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:應用力學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:34
中文關鍵詞:癌細胞爬行微結構孔盤體外模擬化學趨向性
外文關鍵詞:Cell migrationMicrofluidic deviceIn-vitro modelChemotaxis
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體外檢測癌細胞之化學趨向性一直是探討癌症轉移以及癌症惡化的重要研究。在這篇論文中應用微結構孔盤(micro gap plate)來分析量化多種癌症細胞株之侵略能力,由於微結構孔盤可以在每個孔洞中分析單一生長因素對於單一種的癌症細胞株的吸引能力,又因微結構孔盤與市售的96 well孔盤規格相符,所以在每個微結構孔盤中可以有96種不同的分析條件,更可以裝配於顯微鏡的載台上即時觀察,十分符合快速大量的高通量分析方法。本研究提出三種乳癌細胞株(MCF7, MDA-MB-231, HS578T)以及兩種病人取樣細胞株(ABC72PE, ABC37T),本研究提出的乳癌細胞株用細胞特性可以分成上皮表現細胞株(epithelial),上皮-充間質表現細胞株(intermediate epithelial-mesenchymal),充間質表現細胞株(mesenchymal),分別是MCF7, MDA-MB-231, HS578T,而兩種病人取樣細胞株(patient derived cultured cell, PDCC)中,ABC72PE屬於上皮細胞表現(epithelial),ABC37T屬於上皮-充間質細胞表現(intermediate epithelial-mesenchymal)。本研究發現同時具有上皮-充間質細胞表現的細胞株爬行距離以及爬行數量遠超過只具有單一表現(上皮或是充間質)的細胞株,不只有在乳癌細胞株,在更貼近真實病人狀況的病人取樣細胞株(PDCC)也有如此發現,此研究結果可以說明蛋白螢光表現所分別出的癌細胞特性與癌細胞侵略能力是有直接相關,本研究利用高通量之微結構孔盤確實可以做出多種癌細胞化學趨向性高通量之分析,同時也發現同時具有上皮-充間質表現細胞株其侵略能力遠大於單一表現(上皮或是充間質)的細胞株。希望未來可以應用於病人檢體上,幫助醫生提早判斷癌細胞之侵略性以及侵略能力。

In-vitro chemotaxis studies of tumor cells facilitate understanding of tumor progression and assessment of aggressiveness. This work presents the use of a SBS-compatible 96-well plate with special microfluidic gap feature in each well to characterize the aggressiveness of tumor cells with a custom-tailored chemotaxis gradient for each well. This microgap plate (MGP) is also amenable towards imaging under microscopy. The MGP was characterized with three breast cancer cell lines (MCF7, MDA-MB-231 and HS578T) and two types of patient-derived culture cells, or PDCC (ABC72PE and ABC37T). The cell lines are selected based on their phenotypes: epithelial (MCF7), intermediate epithelial-mesenchymal (MDA-MB-231), and mesenchymal (HS578T). The PDCC were cultured from biopsies from metastatic sites of breast cancer patients, with epithelial (ABC72PE) and intermediate epithelial-mesenchymal (ABC37T) phenotypes. Microenvironment with chemotaxis gradients of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) (0, 25, 50 and 100 ng/ml) were tested. Results confirm migration ability of all cell lines exhibit directional movement toward higher EGF/HGF concentration. Further, migration ability of MDA-MB-231 cells with intermediate epithelial-mesenchymal phenotype is more aggressive than either the standalone epithelial (MCF7) or mesenchymal (HS578T) phenotype. Similarly, this intermediate phenotypical characteristic also holds for patient-derived cells where ABC37T exhibits more aggressive behavior than ABC72PE cells. The 96-well high-throughput MGP is shown to be a versatile cellular assay to assess tumor aggressiveness under custom-tailored chemotaxis gradient in each well and might be amenable towards functional assessment of clinical tissues.

1. Introduction 1
2. Material and method 3
2.1 Design concept of Micro-Gap Plate 3
2.2 Growth factor gradient in the MGP 5
2.4 Cell culture and cell lines 7
2.5 Patient derived cultured cells (PDCC) 8
2.6 EMT characterization 10
2.7 Experimental procedure 12
2.7.1 Fabrication and sterilization of MGP 12
2.7.2 Chemotaxis assay preparation and assessment 14
2.8 Statistical analysis 16
3. Results and Discussions 17
3.1 Cell culture in MGP 17
3.2 Effect of different growth factor gradient on cells 19
3.2.1 EGF test 19
3.2.2 HGF test 23
3.2.3 PDCC test 27
3.3 Effect of growth factor on cell morphology 29
4. Conclusions 31
5. Reference 32


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