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국회도서관 홈으로 정보검색 소장정보 검색
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Title Page

Abstract

Contents

1. Introduction 13

2. Materials and Method 21

2.1. Experimental animals and sample collection 21

2.2. Measurement of Protein Half-life 21

2.3. Cell Cultures 22

2.4. Differentiation and Preparation of RNA 22

2.5. Construction of Non-viral and Lentiviral Expression Plasmids 23

2.6. Transfection and Expression test 24

2.7. Large-Scale Transfection and Purification 25

2.8. Lentiviral production and Establishment of THP-1 Polyclone Regulating Luciferase Expression by MFG-E8 Promoter 26

2.9. qRT-PCR Analysis 27

2.10. Cell-Based Screening System. 28

3. Results 29

Part-1. Production of MFGE8 recombinant protein with extended half life 29

3.1. Flow Chart for the Transient Mammalian Expression System 30

3.2. Construction of MFG-E8 Expression Plasmids 32

3.3. Replacement of the MFG-E8 Expression Plasmid with the pCEP4 41

3.4. Comparison of MFG-E8 Protein Expression in pLGNF and pCEP4 Plasmid 44

3.5. Large-Scale Transfection and Purification 47

3.6. Evaluation of Half-Life Extension in Mice 49

3.7. Anti-inflammatory Effect of MFG-E8 Protein in Mice brain 51

Part-2. Establishment of Cell-based assay system for research on MFGE8 protein 55

3.8. Reporter Cell line Development Workflow 56

3.9. Construction of hMFG-E8 Promoter Containing Lenti-Vector 58

3.10. Flow Chart for the Lentiviral Particle and Stable Cell Generation System 63

3.11. PMA-Induced Differentiation of THP-1 Monocyte into Macrophages 65

3.12. Effect of Dexamethasone on Transcriptional Activity of MFG-E8 Promoter in THP-1 Cells 66

3.13. Effect of Single Compounds on Transcriptional Activity of MFG-E8 Promoter. 68

3.14. Single Compounds Treatment Induces an Upregulation of MFG-E8 Expression in THP-1 Cells. 71

3.15. Inhibition of Inflammatory Mediator Expression in THP-1 Cells through Single Compound Treatment 74

4. Discussion 76

5. Reference 80

국문초록 85

List of Tables

Table 1. Nominal half-life values of human proteins in human serum. 20

Table 2. Sequence of the Wild-Type MFG-E8 Expression Plasmid. 35

Table 3. Sequence of the MSA-Fused MFG-E8 Expression Plasmid. 37

Table 4. Features of Engineered Fc. 39

Table 5. Sequence of the IgG-Fc-Fused MFG-E8 Expression Plasmid. 40

Table 6. RT-qPCR Primer Sequence 54

Table 7. Sequence of the hMFG-E8 -1000bp Promoter Region. 62

List of Figures

Figure 1. Sequence Alignment of MFG-E8 in Vertebrates. 17

Figure 2. Domain Structure of MFG-E8 in Vertebrates. 18

Figure 3. Structure and Domain-Specific Functional Mechanisms of Human MFG-E8. 19

Figure 4. Flow Chart of the Transient Mammalian Expression System. 31

Figure 5. Schematics of Various Recombinant MFG-E8 Protein Expression Constructs. 33

Figure 6. Construction of the Wild-Type MFG-E8 Expression Plasmid. 34

Figure 7. Construction of the MSA-Fused MFG-E8 Expression Plasmid. 36

Figure 8. Construction of the hIgG-Fc-Fused MFG-E8 Expression Plasmid. 38

Figure 9. Replacement with pCEP4 Plasmid for Comparative MFG-E8 Protein Expression. 42

Figure 10. Construction of the Wild-Type MFG-E8, MSA-Fused MFG-E8 and hIgG-Fc-Fused MFG-E8 Expression Plasmid. 43

Figure 11. Western Blot Analysis of the Recombinant Human MFG-E8 Protein Expression. 45

Figure 12. Comparison of MFG-E8 Protein Expression in pLGNF and pCEP4 Vectors by Western Blot Analysis. 46

Figure 13. Confirmation of Purified hMFG-E8 Protein. 48

Figure 14. MSA-Conjugated MFGE8 Recombinant Protein Exhibits Elevated Half-Life. 50

Figure 15. Altered Expression of Genes Involved in Inflammation was Found in Cortex of Mice Treated with Recombinant MFGE8 Protein. 52

Figure 16. Altered Expression of Genes Involved in Inflammation was Found in Hippocampus of Mice Treated with Recombinant MFGE8 Protein. 53

Figure 17. Stable Cell Line Generation Workflow Using Lentivirus. 57

Figure 18. Schematics of the Human MFG-E8 Promoter Constructs. 59

Figure 19. Construction of Luciferase Gene Containing Lenti-Vector. 60

Figure 20. Construction of hMFG-E8 Promoter Containing Lenti-Vector. 61

Figure 21. Flow Chart for the Production of Lentiviral Particles and Generation of Stable Cells. 64

Figure 22. PMA-Induced Differentiation of THP-1 Monocyte into Macrophages. 65

Figure 23. Luciferase Activities of hMFG-E8 Promoter were Altered by Treatment of Dexamethasone. 67

Figure 24. Identification of Natural Single Compounds (1-168) that Affect the Activity of hMFG-E8 Promoter Activity. 70

Figure 25. Elevated Expression of hMFG-E8 mRNA was Observed in Differentiated THP-1 Cells Treated with Dexamethasone. 72

Figure 26. The Levels of hMFG-E8 mRNA were Affected by the Treatment with Selected Natural Single Compounds in Differentiated THP-1 Cells. 73

Figure 27. Altered mRNA Expression of Pro-Inflammatory Genes was Observed in Macrophage Cells Treated with Natural Single Compounds. 75

초록보기

 글로벌 의약품 시장은 고령 인구 증가와 만성 질환 증가로 인해 바이오 의약품에 대한 수요가 급증하고 있다. 특정 분자를 표적으로 하는 단백질 기반 약물은 기존 약물에 비해 부작용이 적으나 높은 비용과 짧은 반감기를 극복하는 것은 중요한 연구 과제이다.

본 연구에서는 Milk Fat Globule-EGF Factor 8 (MFG-E8)의 재조합 단백질 생산을 늘리고 생체 내 반감기를 연장하는 전략을 적용하여 생산된 단백질을 평가하였다. 또한, MFGE8 의 상위 조절 인자와 물질을 탐색하기 위한 렌티바이러스 기술을 적용한 세포 기반 스크리닝 시스템을 구축하여 검증하였다.

MFG-E8, MSA-융합 MFG-E8 그리고 Fc-융합 MFG-E8 을 포함한 세 가지 구조적 설계를 개발하여 생산에 영향을 주는 중요 요소를 수정함으로써 단백질 발현의 수준을 평가했다. 이 결과는 발현 벡터의 차이로 인한 발현 수준의 차이로 검증되었다. MSA-융합 MFG-E8 은 무변형 MFG-E8 에 비해 현저히 연장된 반감기를 보였다. 그러나 Fc-융합 MFG-8E 의 한계로 인해 향후 연구에서 구조적 요소 대한 추가적인 조사와 기술적 적용의 필요성이 대두되었다. 마우스 실험에서 복강내 주입된 MFG-E8 은 뇌 조직에서 잠재적인 항염증 반응을 유도하였다. 혈액-뇌 장벽 투과에 대해서는 불확실하지만, 이러한 발견은 단백질에 의한 직, 간접적인 영향의 가능성을 시사한다.

구축된 세포 기반 MFGE8 스크리닝 시스템을 활용하여 염증반응과 연관된 천연물 단일 물질을 성공적으로 스크리닝 선별하였다. 이러한 선행연구를 통하여 향후 MFGE8 의 단백질 의약품으로서 유효성 평가와 MFGE8 을 조절하는 새로운 물질을 탐색하는 연구에 활용할 수 있을 것으로 기대된다.

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