태아연골 유래 줄기세포의 면역 조절에 대한 연구
Immune Modulatory Properties of Human Fetal Cartilage-derived Progenitor Cells
- 주제(키워드) fetal cartilage-derived progenitor cells , immunity , allogeneic lymphocytes , mixed lymphocyte reaction , immunomodulation , acute toxicity , arthritis , inflammation , cell therapy
- 발행기관 아주대학교
- 지도교수 Byoung-Hyun Min
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000020672
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Stem cells can be useful, not only for tissue regeneration but also for inflammatory inhibition and immune modulation effect, etc. Therefore, they are presented as a new therapy for overcoming the limitations of current treatments. For cell injection and transplantation into the human body, the immune responses of cells must be studied exhaustively. Recently, many prior studies researched immune response of MSC. Recently, fetal cartilage-derived progenitor cells (FCPCs) have been described as the promising cell source for tissue engineering and cell therapy. Our previous studies have shown that FCPCs exhibits proliferation and differentiation abilities similar to those of mesenchymal stem cells (MSCs). Many of their characteristics, however, have not been identified yet. Therefore, we investigated immune response of FCPCs in these studies. CHAPTER I: In this study, we investigated in vitro immunogenicity, immune tolerance and immune regulatory function of FCPCs. FCPCs and peripheral blood lymphocytes (PBLs) were isolated from human fetal limbs cartilage and human adult donors, respectively. The PBLs proliferation suppression effect of γ-ray irradiated FCPCs were tested mixed culture by activating PBLs stimulated with Concanavalin A (ConA) as T cell mitogen. This was observed significantly reduction of PBLs proliferation to the 1:1 cell number ratio of FCPCs and PBLs. This result was expected to induce regulatory T cells by cells. Next test was analyzed FoxP3 population as specific marker of regulatory T cell by T cells cultured in the 1:1 cell number ratio of FCPCs and PBLs. Culture of PBLs with FCPCs caused an increase in the number of CD4+T lymphocytes expressing CD25+FoxP3+. The cytokine levels of TNF-α, IFN-γ and IL-10 released were also measured for 4 days of co-culture. The cytokine levels of TNF-α and IFN-γ secreted by stimulation of ConA were steeply increased. However, for PBLs/FCPCs (1:1 or 10:1) co-cultures, there was a significant decrease in TNF-α and IFN-γlevels. The level of IL-10 did not show significant change in cytokine assay. To determine the gene expression of immune-suppressive molecules in FCPCs, FCPCs were treated with IFN-γ and/or TNF-α for 12 hours. We examined no change of TGF-β, LIF, and HLA-G by treatment of pro-inflammatory cytokines. These results suggested that FCPCs act as an immunomodulator and could be used to reduce hypersensitive response of recipients in tissue engineered implants and cell therapies. CHAPTER II: In this study, we investigated the therapeutic potential of human FCPCs by evaluating alleviation of arthritis as well as their local and systemic toxicity. The FCPCs were labeled with PKH-26 and subjected to intra-articular injection in Sprague Dawley (SD) rats. At 1 and 2 weeks after injection, the PKH-26 labeled FCPCs were detected in the synovial membrane, but they were not located in other organs (such as the liver and heart). There were no changes either in the number of white blood cells, red blood cells, and platelets in the hematological analysis or in the weight of the internal organs (such as kidney, spleen, heart and liver) at 1 week. Therapeutic effects on the Complete Freund’s Adjuvant (CFA)-induced knee arthritis in rats were examined by comparing FCPCs results with those of triamcinolone (TRA), a representative anti-arthritis drug. In comparison, that of the TRA-treated group decreased rapidly from day 1, and that of the FCPCs treated group decreased slowly after 7 days. In the histological analysis, both the TRA-treated group and FCPCs-treated group displayed a gradual decrease the infiltration of neutrophils and lymphocytes into the arthritic synovial membrane at 7 and 10 days. These results suggested that FCPCs showed low immune rejection after its injection into the synovial cavity and exerted amelioration of inflammation in rat arthritis model.
more목차
TABLE OF CONTENTS............................................................................................................. i
LIST OF FIGURES.................................................................................................................... iv
LIST OF TABLES...................................................................................................................... vi
ABBREVIATIONS.................................................................................................................... vii
ABSTRACT.............................................................................................................................. viii
Background
1. Stem cell-based therapy.........................................................................................................01
2. Tissue repair by paracrine effect of mesenchymal stem cells (MSCs)...............................02
3. Pitfalls on the autologus and allogenic stem cell therapy...................................................03
4. Immune response of MSCs...................................................................................................04
5. Therapeutic effects of MSCs in arthritis model..................................................................06
6. Characterization of fetus derived tissues and cells.............................................................07
6.1 Fetal cartilage-derived progenitor cells (FCPCs).............................................................09
7. Aim and title of this study......................................................................................................10
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CHAPTER I
Immune Properties of Human Fetal Cartilage-derived Progenitor Cells (FCPCs)
1. Introduction............................................................................................................................12
2. Materials and Methods..........................................................................................................15
2.1 Isolation and culture of human FCPCs...................................................................15
2.2 Surface characterization....................................................................................................15
2.3 Mixed lymphocyte reaction (MLR)..................................................................................16
2.4 Regulatory T cell expression analysis through MLR......................................................16
2.5 Cytokine analysis..............................................................................................................17
2.6 Reverse transcriptase-polymerase chain reaction (RT-PCR)...........................................17
2.7 Statistical analysis.......................................................................................................19
3. Results......................................................................................................................................20
3.1 Immunogenicity of FCPCs...............................................................................................20
3.2 Allogenic response of FCPCs............................................................................................22
3.3 FCPCs suppress mitogen-induced lymphocytes proliferative responses..........................22
3.4 FCPCs increase numbers of regulatory T cells.................................................................24
3.5 FCPCs modulate cytokine production from PBLs............................................................26
3.6 Gene expressions of IDO, LIF, TFG-β, TSG6, sHLA-G, and HGF by FCPCs treated
with IFN-γ and TNF-α........................................................................................................28
4. Discussion................................................................................................................................30
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CHAPTER II
Therapeutic Possibility of Human Fetal Cartilage-derived Progenitor Cells (FCPCs) in Rat Arthritis Model
1. Introduction............................................................................................................................38
2. Materials and Methods..........................................................................................................40
2.1 Isolation and culture of human FCPCs..........................................................................40
2.2 FCPCs distribution analysis in vivo..........................................................................40
2.3 Xenograft study of FCPCs................................................................................................41
2.4 Induction of arthritis animal model and treatments of FCPCs...................................41
2.5 Measurement of the rat knee circumference..................................................................42
2.6 Histological analysis.........................................................................................................42
2.7 Statistical analysis.......................................................................................................42
3. Results......................................................................................................................................44
3.1 FCPCs distribution experiment in vivo......…………………………………………….44
3.2 Autopsy results and organ weights..........................................................................46
3.3 Hematological analysis of post-cells injection.......................................................48
3.4 Inflammatory alleviation effect of FCPCs...............................................................49
3.5 Histological analysis of the synovial membrane of arthritis rats......................................51
4. Discussion................................................................................................................................53
목차
CONCLUSION...........................................................................................................................56
REFERENCE.............................................................................................................................57
국문요약......................................................................................................................................64
