콜히친 반응성에 따른 베체트병 환자에서의 발현 유전자 차이와 Tim3 경로를 차단하는 분자들의 항 종양 효과 비교
Differential gene expression in Behcet's disease and differential anti-tumor effects of Tim3 pathway blocking molecules
- 주제(키워드) Behçet’s disease , colchicine , LPS , IL-6 , TNF-α , C/EBP-β , C/EBP-δ , ATF3 , microRNA , Tim3 pathway , tumor , MDSCs
- 발행기관 아주대학교
- 지도교수 박선
- 발행년도 2013
- 학위수여년월 2013. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000014776
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
PART I Differential expression of genes according to colchicine responsiveness in Behçet’s disease Behçet disease (BD) is a chronic relapsing, systemic inflammatory disorder which is treated with colchicine. However, some patients are not responsive to it. In this study, I wanted to explore the biomarkers to distinguish colchicine responsiveness. I stimulated peripheral blood mononuclear cells (PBMCs) and CD11b+ cells of BD patients with lipopolysaccharide (LPS) and analyzed the expression of inflammatory cytokines, transcription factors and microRNAs. Compared to colchicine-responsive BD patients (BDR), the expression of IL-6 and TNF-α were significantly increased in colchicine-non-responsive BD patients (BDNR) in both the basal and LPS-stimulated states. Concordantly, the expression of C/EBPδ, C/EBPβ transactiption factor of IL-6, was significantly upregulated in BDNR compared to BDR. Also, the expression of ATF-3 transcript was significantly increased in BDR compared to BDNR. The link of dyregulation in these transcription factors to abnormal cytokine production in BD was also demonstrated. Finally, I found the differential expression of miR-638, miR-4488, miR-3591-3p in BD according to colchicine responsiveness. My results suggest that these molecules might be useful to predict colchicine responsiveness in BD.
more초록/요약
PART II Differential anti-tumor effects of three forms of Tim-3 pathway blocking molecules Tim-3 is a molecule containing T cell immunoglobulin variable region (IgV)-like domain and mucin-domain, and known to suppress Th-1 immune response. In a previous study, blocking Tim3 pathway increases anti-tumor immunity leading to tumor growth suppression. In this study, I evaluated the tumor suppressive effect of three different molecules blocking Tim3 pathway: Tim3 IgV like domain alone (Tim3V), fusion protein of Tim3V with mouse immunoglobulin CH2CH3 (Tim3VmIg) and its dimer (Tim3VdIg). Both TimVmIg and Tim3VdIg suppress tumor growth but T3V did not. Compared to control Tim3VdIg but not Tim3V reduced frequency of myeloid derived suppressor cells (MDSCs) in splenocytes of mice on 21 day after tumor challenge. These results indicate that Tim3V domain alone is not sufficient for tumor growth suppression but is in the presence of its fusion partner of immunoglobulin CH2CH3.
more목차
PART I
I. INTRODUCTION 1
A. Behçet’s disease 1
1. Clinical features 1
2. Diagnostic criteria 3
3. Epidemiology 4
4. Etiopathogenesis 5
5. Therapy 10
B. Transcription factors and microRNAs regulating proinflammatory cytokines expression 11
1. Transcription factors 11
2. MicroRNA 12
C. Purpose 13
II. MATERIALS AND METHODS 15
A. MATERIALS 15
1. Subjects 15
2. Culture media 18
3. Reagents 18
4. Antibodies 18
5. Oligonucleotide 19
B. METHODS 19
1. Human peripheral blood mononuclear cells (PBMCs) isolation 19
2. Cell cultures 20
3. CD11b+ cell isolation 20
4. Western blotting 20
5. Lactate dehydrogenase (LDH) release assay 20
6. Enzyme-linked immunosorbent assay (ELISA) 21
7. siRNA transfection 21
8. RNA isolation and cDNA synthesis 21
9. Real-time reverse transcription–polymerase chain reaction (Real-time RT PCR) 21
10. Construction of luciferase reporter vectors 21
11. Luciferase reporter assay 22
12. Statistics 22
III. RESULTS 26
A. Colchicine inhibits caspase-1 activation 26
B. Effect of colchicine on the release of interleukin-1β (IL-1β) is dependent on the inflammatory stimulus and responsiveness to colchicine 28
C. Colchicine does not inhibit lactate dehydrogenase (LDH) release in PBMCs 31
D. Colchicine differentially regulates IL-1β transcription in BD PBMCs according to their colchicines responsiveness 33
E.Transcription factors regulating proinflammatory cytokine production are differentially expressed according to colchicines responsiveness 38
F. The principal cells abnormally expressing cytokines in BDNR are CD11b+ cells 42
G. The knocked-down effect of various transcription factors on expression of TNF-α and IL-6 in THP-1 cells 45
H. The differential effect of siRNA targeting C/EBPβ, C/EBPδ or ATF3 on the cytokine production in CD11b+ cells 48
I. The differential expression of micro RNAs in BD according to colchicine responsiveness 50
IV. DISCUSSION 56
V. CONCLUSION 63
REFERENCES 64
국문요약 79
PART II
I. INTRODUCTION 82
A.The TIM gene family 82
B. Tim3 83
1. Expression 83
2. Structure 83
3. Ligand 84
4. Function in T cell responses 85
5. Roles in tumor immunity 86
C. Roles of myeloid-derived suppressor cells (MDSCs) 87
D. Tumor-associated macrophages (TAMs) 89
E. Purpose 90
II. MATERIALS AND METHODS 91
A. MATERIALS 91
1. Mouse 91
2. Culture media 91
3. Antibodies 91
B. METHODS 92
1. Construction of expression vectors 92
2. Prodcution of recombinant lentiviruses 92
3. Expression of Tim3 pathway blocking molecules 92
4. Estabilishment of stable cells expressing Tim3 pathway blocking molecules 93
5. Measurement of cell growth rate 93
6. Evaluation of tumor growth 94
7. Tumor vaccination 94
8. Flow cytometric analysis of leukocyte subpopulation 94
III. RESULTS 96
A. The characterization of Tim3 pathway blocking molecules 96
B.The tumor growth of 3LL cells stably expressing Tim3 pathway blocking molecules 99
C. The therapeutic effects of 3LL cells stably expressing Tim3 pathway blocking molecules 101
D. The effect of Tim3 pathway blocking molecules on the frequency of MDSCs 103
IV. DISCUSSION 108
V. CONCLUSION 111
REFERENCES 112
국문요약 122
LIST OF TABLES
PART I
Table 1. Smmary of miRNA involvement in the immune response 14
Table 2. The clinical features of BD patients 16
Table 3. Drugs taken by the patients at the time point of bleeding 17
Table 4. Primers for real-time RT PCR 24
Table 5. Sequence of siRNAs 25
Table 6. Primers for CRTC1 3’UTR amplication 25
Table 7. Differential expression of genes related to inflammation according to colchicine responseiveness in Behçet’s disease 55
PART II
Table.1. Primers for cloning of Tim3 pathway blocking molecules 95
Table.2. Primers for detection of transcripts of Tim3 pathway blocking molecules 95
LIST OF FIGURES
PART I
Fig. 1. Diagram of luciferase reporter vectors containing CRTC1 3’UTR proximal and distal region, respectively 23
Fig. 2. Cleavage of caspase-1 is inhibited by colchicine 27
Fig. 3. Effect of colchicine on interleukin (IL)-1β release 30
Fig. 4. Lactate dehydrogenase (LDH) release is not inhibited by colchicine 32
Fig. 5. Effects of colchicine on the transcript levels of interleukin (IL)-1β, tumour necrosis factor (TNF)-a and IL-6 differ in Behc¸et disease peripheral blood mononuclear cells (PBMCs) according to their colchicine responsiveness 35
Fig. 6. The protein levels of TNF-α and IL-6 differ in BD PBMCs according to their colchicine responsiveness 37
Fig. 7. Transcript levels of various transcription factors regulating the proinflammatory cytokine expression 40
Fig. 8. The protein levels of various transcription factors regulating the proinflammatory cytokine expression 41
Fig. 9. The expression of proinflammatory cytokines and their regutory transcription factors in CD11b+ and CD11b- PBMCs 43
Fig. 10. The knock-down effect of various transcription factors on the expression of TNF-α and IL-6 in THP-1 cells 46
Fig. 11. Knockdown of transcription factors differentially affects production of TNF-α and IL-6 in CD11b+ cells according to colchicines responsiveness 49
Fig. 12. The differential expression of micro RNAs in BD PBMCs according to colchicine responsiveness 52
Fig. 13. The miR-4488 mimic oligonucleotide targets distal region of CRTC1 3’UTR 53
Fig. 14. The expression of miR-4488 and its putative target CRTC1 mRNA 54
PART II
Fig. 1. The characterization of Tim3 pathway blocking molecules 98
Fig. 2. The tumor growth in mice challenged with stable cell lines expressing Tim3 pathway blocking molecules 100
Fig. 3. The therapeutic vaccine effects of T3V and T3VdIg in a tumor model 102
Fig. 4. The effects of Tim3 pathway blocking molecules on MDSCs frequency 105
