Studies on the Effects of TNP1 Antigen on Cell-penetration and Pro-inflammatory Cytokine Production Stimulated by AntiDNA Antibodies
- 주제(키워드) TNP1 , Anti-DNA Antibody , Cell-penetration , Lupus Nephritis , Cytokines
- 주제(DDC) 610
- 발행기관 아주대학교
- 지도교수 Young-Ju Jang
- 발행년도 2021
- 학위수여년월 2021. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031038
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Lupus Nephritis (LN), one of the most severe and late-stage manifestation of Systemic Lupus Erythematosus (SLE), is associated with high morbidity and mortality of SLE patients. In LN, the damage of resident renal cells including mesangial cells and deposition of immune complex in glomerular basement membrane with severe kidney inflammation are generally found. Anti-double-stranded DNA (anti-dsDNA) antibodies are associated to the pathogenesis of LN. A subset of anti-DNA antibodies are pathogenic and have cellpenetrating ability. Spermatid Nuclear Transition Protein 1 (TNP1) is one of the potential auto-antigen of LN. I studied the effects of TNP1-synthetic peptide on characteristics of cell-penetrating (2C10, G2-6) and non-cell-penetrating (G5-8) anti-DNA monoclonal antibodies in mouse kidney mesangial (SV40 MES-13) cells, kidney epithelial cells (Renca) and macrophage cells (Raw 264.7). I found that antibodies, 2C10 and G5-8 bound to the TNP1-peptide with higher affinities, but G2-6 with lower affinity by performing the direct-binding and competitive ELISA. It was found that TNP1-peptide induced the cellular changes in the antibody treated cells by performing the flow cytometry, immunofluorescence microscopy and quantitative RT-PCR; the cell-penetration efficiency was reduced by 2C10-TNP1-peptide or G2-6-TNP1-peptide complex as compared to antibody alone. Interestingly, the levels of pro-inflammatory cytokine (interleukin-6 and interferon-α) enhanced by 2C10 or G2-6 or G5-8 in SV40 MES-13 cells were decreased when treated with antibody-TNP1-peptide complex, whereas those were increased in Renca and Raw 264.7 cells. My results indicate that TNP1-peptide has ability to reduce the cell-penetration efficiency of anti-DNA antibody, and acts in differently among various cell lines in relation to pro-inflammatory cytokine expression which could be of potential therapeutic targets in LN. These results suggest, TNP1-peptide has a potential therapeutic significance in reducing the inflammatory condition by blocking the cell-penetrating and pathogenic activity of anti-DNA antibodies.
more목차
I. INTRODUCTION 1
II. OBJECTIVES 4
A. General 4
B. Specific 4
III. MATERIALS AND METHODS 5
A. Experimental Cell Lines 5
B. Anti-DNA mAb (monoclonal Antibody) 5
C. Spermatid Nuclear Transition Protein 1 (TNP1) 5
D. Cytokine Primers 5
E. Total RNA extraction and cDNA synthesis 6
F. Enzyme-Linked Immunosorbent Assay (ELISA) 6
1. Direct binding ELISA 7
2. Direct binding ELISA for TNP1-peptide 7
3. Inhibition ELISA for TNP1-peptide 7
G. Immunofluorescence Microscopy (IFM) 8
H. Flow cytometry 8
I. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 9
J. Statistical Analysis 9
IV. RESULTS 10
A. ELISA 10
1. ds-DNA direct binding ELISA 10
2. ss-DNA direct binding ELISA 10
3. TNP1-peptide direct binding ELISA 11
4. TNP1-peptide inhibition ELISA 11
B. Flow cytometry 14
C. IFM 18
1. IFM of mAbs on time dependent 18
2. IFM with mAb and TNP1-peptide 18
D. qRT-PCR 24
1. Time-dependent cytokine mRNA expression levels 24
2. TNP1-peptide alone effects on cytokine expression 24
3. Effects of TNP1-peptide and mAb complex on cytokine mRNA expression 25
V. DISCUSSION 38
VI. CONCLUSION 43
VII. RECOMMENDATION 44
REFERENCES 45
