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Graduate School of Ajou University Department of Biomedical Sciences 3. Theses(Master)

콜히친 반응성에 따른 베체트병 환자에서의 발현 유전자 차이와 Tim3 경로를 차단하는 분자들의 항 종양 효과 비교

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dc.contributor.advisor박선-
dc.contributor.authorWoo, Min Yeong-
dc.date.accessioned2022-11-29T03:01:06Z-
dc.date.available2022-11-29T03:01:06Z-
dc.date.issued2013-08-
dc.identifier.other14776-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/20740-
dc.description학위논문(박사)아주대학교 일반대학원 :의생명학과,2013. 8-
dc.description.tableofcontentsPART 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-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title콜히친 반응성에 따른 베체트병 환자에서의 발현 유전자 차이와 Tim3 경로를 차단하는 분자들의 항 종양 효과 비교-
dc.title.alternativeMin-yeong Woo-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameMin-yeong Woo-
dc.contributor.department일반대학원 의생명과학과-
dc.date.awarded2013. 8-
dc.description.degreeMaster-
dc.identifier.localId571234-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000014776-
dc.description.alternativeAbstractPART 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.-
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