나노 섬유 지지체내에서 3차원적 세균탐식과 탐식세포 이동 분석
Bacterial infection-mimicking three-dimensional phagocytosis and chemotaxis in nanofibrous scaffold
- 주제(키워드) 3D cell culture , phagocytosis , migration , S.aureus
- 발행기관 아주대학교
- 지도교수 곽종영
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000025502
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Professional phagocytes such as neutrophils, macrophages, and dendritic cells (DCs) actively engulf microbes. In this study, we developed a three-dimensional (3D) in vitro infection model for investigating the cross-talk between phagocytes and microbes in inflammation. A culture system using a nanofibrous scaffold (NFS) having multi-planar pores in a 3D structure was used for coculture of Staphylococcus aureus (S.aureus) and phagocytes in the same as well as different compartments. Surface-seeded S. aureus and phagocytes were able to adhere to nanofibers inside the NFS and phagocytes migrated to and engulfed S. aureus in a 3D manner. The addition of formyl peptide receptor antagonists decreased the phagocytic rate in 3D NFS but not in two-dimensional (2D) culture dishes. The migration of phagocytes to S. aureus was evaluated in an NFS-based layer-by-layer culture system. Neutrophils, macrophages, and DCs cultured in an upper NFS migrated to the lower NFS containing bacteria. Cytokine and chemokine secretion patterns by neutrophils cultured with S. aureus differed between 2D and 3D culture conditions. DCs migrated to neutrophils phagocytosing bacteria and then engulfed neutrophils in 3D in vitro culture. In addition, neutrophils and macrophages in the upper NFS migrated to bacteria-infected lung epithelial cells in the lower NFS of the layer-by-layer system. S. aureus-infected lung epithelial cells stimulated the secretion of tumor necrosis factor (TNF)-α and IL-1ain 3D culture condition, but not in 2D culture. Therefore, NFS-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo.
more목차
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIAL AND METHODS 4
A. Materials 4
B. Electrospinning and fabrication of the PCL nanofibers 4
C. Preparation of phagocytes from bone marrow 5
D. Preparation of peritoneal neutrophils and macrophages 6
E. MLE-12 cell culture 6
F. Bacterial culture and adhesion assay 6
G. Laser confocal microscopy 7
H. Scanning electron microscopy (SEM) 7
I. FACS analysis of phagocytosis 7
J. Live imaging of phagocytosis 8
K. Migration assay in NFS-based layer-by-layer system 9
L. Cytokine array analysis 9
M. Statistical analysis 10
Ⅲ. RESULTS 11
A. 3D culture of S. aureus in electrospun PCL NFS 11
B. Engulfment of S. aureus by phagocytes in the NFS 13
C. Differential rate of phagocytosis in 2D and 3D culture conditions 15
D. 3D migration of phagocytes to S. aureus in NFS-based layer-by-layer culture system 20
E. NFS-based horizontal system 23
F. Neutrophil-induced recruitment of more neutrophils to S. aureus in NFS-based layer-by-layer culture system 25
G. Engulfment of neutrophils by DCs in the presence of S. aureus in 2D and 3D culture conditions 30
H. Differential secretion of cytokines and chemokines by neutrophils cultured with S. aureus in 2D versus 3D culture conditions 32
I. Migration of phagocytes to S. aureus-infected epithelial cells in NFS-based layer-by-layer system 35
Ⅳ. DISCUSSION 39
Ⅴ. CONCLUSION 43
REFERENCE 44
국문요약 48
