폐암 주변 미세환경을 모사한 하이드로젤에서 마이크로플루이딕 3차원적 폐암세포 배양

학위논문 (석사)-- 서울대학교 대학원 : 재료공학부, 2016. 8. 안철희.In vitro, lung cancer has been studied in 2D, tissue culture plate by many cancer researchers. Recently, changes of study trend in microenvironment have moved 2D to 3D. Many papers have reported differences of results in 2D culture and 3D culture. The in vitro results in 3D culture were more similar to in vivo results than those in 2D. Thus, many researchers have studied cells using hydrogels. One of the hydrogels is collagen type Ⅰ that is a most common extracellular matrix (ECM). However, the composition of ECM in each tissue is different due to the different function. Lungs are organs in respiratory system, and those have elasticity for extension and contraction. The ECM in lungs has known that collagen and elastin are the major components. Our study focused on lung microenvironment, and using microfluidic devices we demonstrated lung cancer cells in hydrogels mimicking lung ECM which are composed of collagen type Ⅰ, collagen type Ⅲ, and elastin (CCE). These results have shown that differences of morphologies and bioactivities in RNA levels, protein levels, and enzyme levels are remarkable. Furthermore, our studies suggest that difference of hydrogel property could affect responses of lung cancer cells.1. Introduction 1 2. Experiments 3 2.1. Materials 3 2.2. Fabrication of microfluidic device 4 2.3. Gelation of hydrogels 6 2.4. Cell culture in 2D condition 6 2.5. Cell culture in microfluidic devices 7 2.6. Cell proliferation assay (CCK-8) 7 2.7. Zymography analysis of cells 8 2.8. Western blot analysis of cells 9 2.9. RNA extraction from tissues and cDNA synthesis 10 2.10. Real-time PCR 11 2.11. Immunofluorescence staining 13 2.12. Live cell imaging 13 3. Results and Discussion 15 3.1. Difference of CD44 expression of A549 cells according to the hydrogels 15 3.2. Proliferation analysis of A549 cells 19 3.3. A549 cells in 3D hydrogels produce Matrix Metalloproteinases not produced in 2D culture 21 3.4. Marked differences in RNA levels 23 3.5. Noticeable results in Protein levels 25 3.6. Response of A549 cells in CCE gels 27 4. Conclusions 29 5. References 30 요약문 33Maste