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국회도서관 홈으로 정보검색 소장정보 검색
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결과 내 검색 동의어 포함

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Title Page

Contents

ABSTRACT 6

INTRODUCTION 8

1. Atherosclerosis 8

2. Macrophage-specific promoters 15

3. Adiponectin, anti-atherogenic gene 17

4. Objective 18

PART 1 : DEVELOPMENT OF A MACROPHAGE-SPECIFIC SYNTHETIC PROMOTER 19

ABSTRACT 19

INTRODUCTION 20

1. Macrophage-specific promoters and elements 20

2. P47phox promoter and PU.1 binding site(이미지참조) 23

3. Objective 24

MATERIALS AND METHODS 25

1. Synthetic promoters 25

2. Cell culture and transient transfection 25

3. Luciferase assay 26

4. Statistical analysis 26

RESULTS 27

1. Synthetic promoter 27

2. Macrophage-specific activities of SP107 and SP146 30

3. Modification of synthetic promoter 34

4. Macrophage-specific activities of modified synthetic promoters 39

DISCUSSION 43

PART 2 : EFFECT OF SYNTHETIC PROMOTER ON ATHEROCLEOTIC CONDITIONED CELLS 46

ABSTRACT 46

INTRODUCTION 47

1. Roles of macrophage in atherosclerosis lesion 47

2. Adiponectin in atherosclerosis 48

3. Objective 50

MATERIALS AND METHODS 51

1. Construction of plasmids 51

2. Cell culture 51

3. Reverse transcriptase-polymerase chain reaction (RT-PCR) 52

4. Western blot 53

5. Luciferase assay 53

6. Foam cell formation assay 53

7. Statistical analysis 54

RESULTS 55

1. Construction of adiponectin expression vector 55

2. Expression test of adiponectin in cell lines 57

3. Construction of macrophage-specific adipoectin expression plasmid vector 59

4. Macrophage-specific adiponectin expression test in various cell lines 62

5. Anti-atherogenic effect of SP-APN on foam cell formation 65

6. Construction of SP-APN lentiviral vector and macrophage specificity 68

7. Anti-atherogenic effect of SP-APN lentivirus on foam cell formation 73

DISCUSSION 76

PART 3 : THERAPEUTIC APPLICATION OF MACROPHAGE-SPECIFIC SYNTHETIC PROMOTER IN AN ATHEROSCLEROSIS ANIMAL MODEL 79

ABSTRACT 79

INTRODUCTION 81

1. Atherosclerosis animal models 81

2. Objective 83

MATERIALS AND METHODS 84

1. Animals and subtotal occlusion model 84

2. Lipid staining in plaque 84

3. Immunohistochemical analysis 85

4. Statistical analysis 85

RESULTS 86

1. Induction of atherosclerosis animal model by subtotal occlusion on left carotid artery 86

2. Subtotal occlusion in ApoE KO mice 89

3. Appication of lenti-SP-APN for one week on subtotal occlusion model 91

4. Appication of lenti-SP-APN for two week on subtotal occlusion model 94

DISCUSSION 98

SUMMARY 100

REFERENCES 102

국문초록 115

초록보기

배경 : 대식세포의 거품세포 형성은 동맥경화를 유발하는 주요한 원인으로 보고 되었다. 많은 연구들은 대식세포에 특이적인 프로모터를 보고하였지만 치료용 벡터로 활용하기에는 특이성 및 효율성이 충분하지 않았다. 본 연구에서는 저자가 개발한 대식세포 특이적 합성 프로모터에 adiponectin 유전자를 연결하여 동물 동맥경화 모델에 적용하여 그 효과를 검증하고자 하였다.

방법 : 합성 프로모터 SP-146은 NCBI 웹 사이트[http://www.ncbi.nim.nih.gov/nuccore/DQ107383]에 등록되어 있으며, 비 대식 세포 세포주 (293T, HeLa)와 대식세포 세포주(RAW264.7)에 도입하여 프로모터 활성을 분석하였다. 대식세포 특이성을 증진시키기 위하여 PU.1 인자를 포함하는 p47phox 프로모터의 일부분을 다양한 길이(C1, C2, C3)로 제작하여 합성 프로 모터 뒤에 삽입하였다. RAW264.7 세포에서 분석했을 때, 후보 프로모터들 사이에서 SP146-C1 이 가장 높은 특이성과 효율을 보였고, 최종적으로 이 프로모터가 대식세포 특이적 adiponectin 발현 벡터의 개발에 선택되었다. C1 부위의 PU.1 과 전사 시작 부위 사이의 정확한 서열의 길이가 세포 특이적 활성에 중요한 인자로 여겨진다. Adiponectin 유전자를 치료적 유전자로 선택하여 SP146-C1 vector에 삽입하였다.

결과 : 다양한 세포에서 adiponectin의 특이적 발현을 확인하였고 거품세포 형성 분석을 통해 adiponectin의 기능을 검증하였다. SP146-C1 프로모터의 발현 효율은 CMV 프로모터에 비할 만 하였다. 그러나 플라스미드를 이용한 유전자 치료는 낮은 유전자 도입 효율과 짧은 발현 시간으로 인한 약점을 가지고 있다. 그래서 SP146-C1에 의해 조절되는 GFP 또는 adiponectin 렌티 바이러스를 제작하였고 세포 특이성을 검증하였다. 거품세포 형성을 유도하기 위하여 RAW264.7 세포에 산화된 저밀도 지단백(oxidized LDL, 100㎍/mL)을 1일간 처리하였다. 세포에 축적된 지방의 수준은 Oil red O 염색으로 분석하였다. adiponectin의 과발현은 성공적으로 RAW264.7 세포의 지방 축적을 억제시켰다. 동맥경화반에 대식세포 특이적 adiponectin의 발현이 치료적 효과를 갖는지 검증하기 위하여, ApoE KO 쥐에 partial carotid ligation 수술을 통해 동맥경화를 유발하였고, jugular vein을 통해 SP-APN 렌티바이러스를 투여하였다. SP-APN 바이러스를 처리한 쥐에서 동맥경화반의 크기와 지방 축적 수준이 유의적으로 감소하였고, 대식세포에 특이적으로 adiponectin이 발현하는 것을 확인하였다.

결론 : SP146-C1 프로모터는 P47phox 프로모터와 합성 프로모터 SP146의 조합으로 만들어졌으며 성공적으로 대식세포를 표적함을 증명하였고, 이를 통한 adiponectin의 발현 조절은 동맥경화증의 발생을 억제하였다.

참고문헌 (99건) : 자료제공( 네이버학술정보 )

참고문헌 목록에 대한 테이블로 번호, 참고문헌, 국회도서관 소장유무로 구성되어 있습니다.
번호 참고문헌 국회도서관 소장유무
1 Macrophages in the Pathogenesis of Atherosclerosis 네이버 미소장
2 The response-to-retention hypothesis of early atherogenesis. 네이버 미소장
3 Monocyte-Endothelial Cell Interactions in the Development of Atherosclerosis 네이버 미소장
4 ATP-Binding Cassette Transporters and HDL Suppress Hematopoietic Stem Cell Proliferation 네이버 미소장
5 Leukocytosis and ischemic vascular disease morbidity and mortality: is it time to intervene? 네이버 미소장
6 Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis 네이버 미소장
7 Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. 네이버 미소장
8 Macrophage heterogeneity in atherosclerotic plaques 네이버 미소장
9 Identification of a Novel Macrophage Phenotype That Develops in Response to Atherogenic Phospholipids via Nrf2 네이버 미소장
10 Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. 네이버 미소장
11 Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation. 네이버 미소장
12 Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. 네이버 미소장
13 Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages. 네이버 미소장
14 The macrophage cholesterol exporter ABCA1 functions as an anti-inflammatory receptor. 네이버 미소장
15 CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation. 네이버 미소장
16 NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. 네이버 미소장
17 Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. 네이버 미소장
18 Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western diet-fed apolipoprotein E-deficient mice 네이버 미소장
19 Regulation of macrophage function in inflammation and atherosclerosis. 네이버 미소장
20 The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages 네이버 미소장
21 Atherogenic Lipids and Lipoproteins Trigger CD36-TLR2-Dependent Apoptosis in Macrophages Undergoing Endoplasmic Reticulum Stress 네이버 미소장
22 Consequences and Therapeutic Implications of Macrophage Apoptosis in Atherosclerosis: The Importance of Lesion Stage and Phagocytic Efficiency 네이버 미소장
23 Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses. 네이버 미소장
24 Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR 네이버 미소장
25 The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques. 네이버 미소장
26 Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention. 네이버 미소장
27 Laser Capture Microdissection Analysis of Gene Expression in Macrophages from Atherosclerotic Lesions of Apolipoprotein E-Deficient Mice 네이버 미소장
28 Macrophages in atherosclerosis: a dynamic balance. 네이버 미소장
29 Macrophage-Specific Gene Expression: Current Paradigms and Future Challenges 네이버 미소장
30 The macrosialin promoter directs high levels of transcriptional activity in macrophages dependent on combinatorial interactions between PU.1 and c-Jun. 네이버 미소장
31 Scavenger Receptor A Gene Regulatory Elements Target Gene Expression to Macrophages and to Foam Cells of Atherosclerotic Lesions 네이버 미소장
32 Development of a Synthetic Promoter for Macrophage Gene Therapy 네이버 미소장
33 Evaluation of macrophage-specific promoters using lentiviral delivery in mice. 네이버 미소장
34 A novel serum protein similar to C1q, produced exclusively in adipocytes. 네이버 미소장
35 Proteolytic Cleavage Product of 30-kDa Adipocyte Complement-Related Protein Increases Fatty Acid Oxidation in Muscle and Causes Weight Loss in Mice 네이버 미소장
36 The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity 네이버 미소장
37 Vascular effects of adiponectin: molecular mechanisms and potential therapeutic intervention. 네이버 미소장
38 Adiponectin – journey from an adipocyte secretory protein to biomarker of the metabolic syndrome 네이버 미소장
39 Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. 네이버 미소장
40 Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell. 네이버 미소장
41 Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. 네이버 미소장
42 Disruption of adiponectin causes insulin resistance and neointimal formation. 네이버 미소장
43 Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. 네이버 미소장
44 Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. 네이버 미소장
45 Absence of Monocyte Chemoattractant Protein-1 Reduces Atherosclerosis in Low Density Lipoprotein Receptor–Deficient Mice 네이버 미소장
46 Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice. 네이버 미소장
47 Retrovirus-mediated expression of apolipoprotein A-I in the macrophage protects against atherosclerosis in vivo. 네이버 미소장
48 Long-Term Stable Expression of Human Apolipoprotein A-I Mediated by Helper-Dependent Adenovirus Gene Transfer Inhibits Atherosclerosis Progression and Remodels Atherosclerotic Plaques in a Mouse Model of Familial Hypercholesterolemia 네이버 미소장
49 Current developments in the design of onco-retrovirus and lentivirus vector systems for hematopoietic cell gene therapy. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2003; 1640:1-24. 미소장
50 Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro. 네이버 미소장
51 Fine Structure Genetic Analysis of a β-Globin Promoter 네이버 미소장
52 Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. 네이버 미소장
53 Distinct Regions of Sp1 Modulate DNA Binding and Transcriptional Activation 네이버 미소장
54 Myeloid-specific gene expression. 네이버 미소장
55 Transcription factors, normal myeloid development, and leukemia. 네이버 미소장
56 Improving safety of gene therapy. 네이버 미소장
57 PU.1 is essential for p47(phox) promoter activity in myeloid cells. 네이버 미소장
58 Critical flanking sequences of PU.1 binding sites in myeloid-specific promoters. 네이버 미소장
59 Adipose tissue dysfunction in obesity, diabetes, and vascular diseases 네이버 미소장
60 Globular adiponectin upregulates nitric oxide production in vascular endothelial cells 네이버 미소장
61 Adiponectin stimulates production of nitric oxide in vascular endothelial cells. 네이버 미소장
62 Adipocyte-Derived Plasma Protein, Adiponectin, Suppresses Lipid Accumulation and Class A Scavenger Receptor Expression in Human Monocyte-Derived Macrophages 네이버 미소장
63 The NADPH oxidase and chronic granulomatous disease 네이버 미소장
64 Characterization of the 47-kilodalton autosomal chronic granulomatous disease protein: tissue-specific expression and transcriptional control by retinoic acid. 네이버 미소장
65 Induction of expression of genes encoding components of the respiratory burst oxidase during differentiation of human myeloid cell lines induced by tumor necrosis factor and gamma-interferon. 네이버 미소장
66 Expression of p47-phox and p67-phox proteins in murine bone marrow-derived macrophages: enhancement by lipopolysaccharide and tumor necrosis factor alpha but not colony stimulating factor 1. 네이버 미소장
67 Suppression of superoxide anion production by interleukin-10 is accompanied by a downregulation of the genes for subunit proteins of NADPH oxidase. 네이버 미소장
68 Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components. 네이버 미소장
69 Cooperative effects of interferon-gamma on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells. 네이버 미소장
70 The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene 네이버 미소장
71 The Ets family of transcription factors 네이버 미소장
72 Terminal myeloid gene expression and differentiation requires the transcription factor PU.1. 네이버 미소장
73 New insights on DNA recognition by ets proteins from the crystal structure of the PU.1 ETS domain-DNA complex. 네이버 미소장
74 A new pattern for helix-turn-helix recognition revealed by the PU.1 ETS-domain-DNA complex. 네이버 미소장
75 Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. 네이버 미소장
76 Expression of two myeloid cell-specific genes requires the novel transcription factor, c-fes expression factor. 네이버 미소장
77 PU.1 as an Essential Activator for the Expression of gp91phoxGene in Human Peripheral Neutrophils, Monocytes, and B Lymphocytes 네이버 미소장
78 The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. 네이버 미소장
79 PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. 네이버 미소장
80 Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor. 네이버 미소장
81 Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif. 네이버 미소장
82 The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter. 네이버 미소장
83 CD18 (beta 2 leukocyte integrin) promoter requires PU.1 transcription factor for myeloid activity. 네이버 미소장
84 Improving safety of gene therapy. 네이버 미소장
85 Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. 네이버 미소장
86 Adiponectin: protection of the endothelium. Curr Diab Rep. 2005; 5:254-9. 미소장
87 Cyclic Cushing's syndrome: a clinical challenge. 네이버 미소장
88 Inverse relationship between adiponectin levels and subclinical carotid atherosclerosis in patients undergoing coronary artery bypass grafting. 네이버 미소장
89 Decreased plasma adiponectin concentrations in nondiabetic women with elevated homeostasis model assessment ratios. 네이버 미소장
90 Reciprocal Association of C-Reactive Protein With Adiponectin in Blood Stream and Adipose Tissue 네이버 미소장
91 Circulating Adipocyte-Fatty Acid Binding Protein Levels Predict the Development of the Metabolic Syndrome: A 5-Year Prospective Study 네이버 미소장
92 Lipocalin-2 is an inflammatory marker closely associated with obesity, insulin resistance, and hyperglycemia in humans. 네이버 미소장
93 Pioglitazone ameliorates insulin resistance and diabetes by both adiponectin-dependent and -independent pathways. 네이버 미소장
94 Hypoadiponectinemia is closely linked to endothelial dysfunction in man. 네이버 미소장
95 Flow-Induced Vascular Remodeling in the Mouse 네이버 미소장
96 Angiotensin II–Induced Hypertension Accelerates the Development of Atherosclerosis in ApoE-Deficient Mice 네이버 미소장
97 Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. 네이버 미소장
98 Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis. 네이버 미소장
99 Bone morphogenic protein antagonists are coexpressed with bone morphogenic protein 4 in endothelial cells exposed to unstable flow in vitro in mouse aortas and in human coronary arteries: role of bone morphogenic protein antagonists in inflammation and atherosclerosis. 네이버 미소장

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