축삭재생 촉진에 관여하는 대식세포의 분자적 특성
Molecular determinants of macrophages with a
- 주제(키워드) Axon regeneration , macrophage , RAM , TAM
- 발행기관 아주대학교
- 지도교수 김병곤
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027955
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Although intrinsic capacity of CNS axon regeneration is highly limited, injuries to the peripheral nerves frequently result in successful axon regeneration. We have previously shown that macrophage activation in the dorsal root ganglia (DRGs) following peripheral nerve injury contributes to the enhanced axon regeneration capacity. We further demonstrated that neuron-macrophage interaction occurring in the DRGs is essential linking injury-triggered neuronal chemokine expression with the proregenerative macrophage activation. However, it is not known how activated macrophages, which we defined as regeneration-associated macrophages (RAMs), in turn contribute to the enhanced capacity of axon regeneration in DRG neurons. The present study sought to characterize molecular signatures of RAMs and to identify key signaling pathways that drive the activation of the proregenerative phenotype in RAMs. To generate RAMs, we employed an in vitro neuron-macrophage interaction model where co-cultures of DRG neurons and peritoneal macrophages are treated with cAMP. In this model, conditioned medium of primed macrophages promotes robust neurite outgrowth. We performed transcriptomic profiling using RAMs obtained in this co-culture paradigm. Ingenuity Pathway Analysis indicated that genes related to cancer and wound healing processes, which are known to increase in tumor-associated macrophages, were highly upregulated in RAMs. Furthermore, analysis of upstream transcriptional networks suggested that beta-catenin and Hif1-alpha could play a role in shaping the transcriptomic signatures. To verify the upregulated genes in an in vivo neuron-macrophage interaction setting, CX3CR1-GFP mice were utilized to isolate activated macrophages in DRGs following peripheral nerve injury using Fluorescence-Activating Cell Sorting (FACS). Ongoing experiments involving analysis of the FACS-isolated macrophages with immunohistochemistry and real time PCR will characterize the molecular determinants of RAMs promoting capacity of axon regeneration in vivo.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURE v
ABBREVIATION vi
I. INTRODUCTION
A. Failure in spontaneous axon regeneration in CNS neurons 1
B. Successful axon regeneration model: conditioning injury 2
C. Neuron-Macrophage interaction in conditioning injury paradigm 3
D. Spectrum of macrophage phenotype 5
E. Aims of the thesis research 6
II. Materials and methods
1. Animals and Surgical procedures 7
2. Neuron-macrophage co-culture system 7
3. RNA sequencing and Ingenuity Pathway Analysis (IPA) 8
4. Fluorescence-Activating Cell Sorting (FACS) for isolation of macrophages 9
5. Quantitative reverse transcription polymerase chain reaction 10
6. Tissue processing and immunofluorescence staining 11
7. Statistical analysis 11
III. RESULTS
1. Gene expression profiling in RAMs generated by in vitro neuron-macrophage interaction model 15
2. Gene expression network analysis 20
3. Validation of gene expression profiling in vivo using CX3CR1-GFP mice 25
4. Activation of Wnt/β-catenin pathway in perineuronal macrophages in DRGs following SNI 30
IV. DISCUSSION 34
V. CONCLUSION 38
REFERENCE 39
국문요약 45
