臺灣博碩士論文加值系統

根據統計結果，癌症在過去一直是全球十大死因的疾病之一，而造成癌症死亡的主要原因便是癌細胞擴散，臨床的紀錄顯示，當原位癌開始轉移擴散至身體其他組織器官時，病人的存活率有著非常顯著的下降；然而關於癌症轉移方面的了解卻非常有限，原因有：沒有一個適當的平台能夠在體外重建癌症生長的環境進而觀察癌症轉移的現象、動物實驗非常昂貴且觀察不易。所以本論文致力於利用微流體的技術建置一微流晶片，在晶片中模擬癌細胞生長的環境，使癌細胞能夠在此晶片中完成初步的轉移，並能夠對此轉移現象進行長時間且及時的觀察及數據統計。
本實驗使用了兩種不同類型的人類乳癌細胞(MCF7，MDA-MB-231)，並且進一步將癌細胞培養呈球形以求更接近癌症在體內的真實形體來進行實驗，在外型上MDA-MB-231的癌細胞是屬於狹長型細胞，相較於MCF7是具備較高轉移能力的癌細胞類型；而在實驗觀測上是以癌細胞轉移的速率及顆數來量化轉移的能力，結果顯示MDA-MB-231確實比MCF7具備了更高的轉移能力，符合預期中的結果，且曾經在晶片中對轉移的癌細胞進行長時間(14天)的細胞培養，證明了此晶片所建置的環境是非常適合癌細胞生長的環境，也證明此晶片具備了進行完整癌症轉移現象觀察的潛力。而在觀察癌症轉移的同時，次晶片亦可同時進行癌症的化療藥物測試，以求能夠篩選出能夠治療癌症且同時能夠抑制癌症轉移的化療藥物，期望未來臨床上能夠提供醫師進行癌症化療時用藥的參考。

In vitro studies of the complex tumor microenvironment facilitate understanding of tumor progression and even drug efficacy. In this study, 3D tumor metastasis and associated anti-cancer drug screening were studied in a microfluidic chip. The metastatic process was modeled by cells extravasate from spheroids composed of breast cancer cell lines (MCF7 and MDA-MB-231). These cells then migrated in response to EGF chemotaxi gradient with and without the influence of anti-cancer drugs.
The chip consists of two parallel top channels and a bottom channel sandwiching a perforated membrane patterned with 50 μm holes. Solution exchange was driven by the pressure gradient caused by different liquid surface level. Herein, an evaporation-driven pump (EDP) was used to replace the traditional pump to maintain the concentration gradient during the experiment.
The migration ability of tumor spheroid had been compared to the parental single cell and results showed that tumor spheroid had an upregulated EGFR expression and a higher invasive ability than parental cells. Also, an anti-cancer drugs, paclitaxel, were introduced to do the drug test on tumor spheroids. The results showed that the IC50 value of tumor spheroid is larger than parental cell, suggesting that tumor spheroid acquire better drug resistance than parental cells. Furthermore, paclitaxel can also be served as an anti-migratory drug so we can obtain the inhibition of cancer metastasis result at the same time.
This device enables study of 3D and 2D cell metastases and associated drug screening on tumor spheroid and might be useful for clinical study of personalized therapy.

致謝……………………………………………………………………………………...i
中文摘要………………………………………………………………………………..ii
ABSTRACT…………………………………………………………………………….iii
CONTENTS…………………………………………………………………………….iv
LIST of FIGURES……………………………………………………………………...vi
LIST of TABLES……………………………………………………………………….vii
1. Introduction………………………………………………………………………….1
2. Material and Method………………………………………………………………..3
2.1 Design concept of the microfluidic device……………………………………….3
2.2 Evaporation-driven pump………………………………………………………..6
2.3 Chip Fabrication………………………………………………………………….6
2.4 Cell lines………………………………………………………………………….7
2.5 Spheroid culture………………………………………………………………….7
2.6 Chemosensitivity assay…………………………………………………………..8
2.7 Evaluation of tumor spheroid size………………………………………………..8
2.8 Experiment procedure……………………………………………………………8
3. Results and Discussion……………………………………………………………..10
3.1 Characterization of evaporation-driven pump and the concentration gradient
generator…………………………………………………………………………10
3.2 In vitro microfluidic chemotaxis assay of 3D spheroids………………………..12
3.2.1 Monolayer v.s. spheroid cells migration assay……………………………13
3.2.2 Effect of different EGF concentration on tumor cell migration…………..15
3.2.3 Effect of different spheroid size on tumor cell migration…………………17
3.3 Anti-cancer drug screening………………………………………………………19
3.3.1 Morphology of cells………………………………………………………..21
3.3.2 Drug toxicity profiles in the chip…………………………………………..23
4. Conclusion…………………………………………………………………………..24
Appendix……………………………………………………………………………….25
Biomimetic nano-cilia generate multicellular tumor spheroids……………………….25
Experimental methods to study tumor cell migration………………………………….29
Reference……………………………………………………………………………….32

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