TNF-α와 IL-1β로 자극 된 태아 연골 유래 줄기 세포에 의한 활막 세포 염증 마커의 하향 조절
TNF-α and IL-1β challenged fetal cartilage-derived stem cells results in down-regulation of inflammatory markers in synoviocytes
- 주제(키워드) stem cell , priming , anti-inflammatory effect
- 발행기관 아주대학교
- 지도교수 민병현
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028844
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Abstract PURPOSE. Fetal cartilage-derived stem cells (FCSCs) have been shown to contribute to OA therapy as novel cells with anti-inflammatory properties. However, efforts to enhance the anti-inflammatory ability of FCSCs have not yet been conducted. In this study, the anti-inflammatory properties of FCSCs were increased by priming and evaluated in inflammatory synoviocytes. METHODS. FCSCs were isolated from the fetal cartilage tissue. Priming was carried out by stimulating FCSCs using a priming factor such as Poly(I:C), IFN-γ, TNF-α and IL-1β and cell culture supernatants were obtained as conditioned media (CM). The anti-inflammatory effects of FCSCs were evaluated by treating CM in inflammatory synoviocytes. The evaluation was carried out by confirming the expression of the pro-inflammatory gene TNF-α, IL-1β and IL-6 through qRT-PCR. In addition, the concentration of PGE2 secreted by inflammatory synoviocytes was measured through ELISA. To identify the factors that affect the anti-inflammatory effects of FCSCs, the growth factors (IGF-1, TGF-β and FGF-2) were measured with qRT-PCR and the expression of IDO-1 was confirmed with western blotting. We also identified various secreted factors in the CM of FCSCs through Protein Array. Finally, we analyzed the characteristics of FCSCs using a Microscope, FACS and Histologic analysis to confirm that the stem cell characters are maintained. RESULTS. When TNF-α (10 ng/mL) and IL-1β (15 ng/mL) were treated with FCSCs for 24 h, the anti-inflammatory properties of FCSCs were most significantly increased. In inflammatory synoviocytes treated with CM of primed-FCSCs, the expression of the pro-inflammatory gene TNF-α, IL-1β and IL-6 decreased. In addition, the concentration of PGE2 was significantly reduced in CM of primed-FCSCs. The anti-inflammatory effects of FCSCs were maximized when the concentration of CM was doubled. The expression of IGF-1, TGF-β, FGF-2 and IDO-1 significantly increased after priming, and the Protein Array confirmed an increase in the secretion of more than 20 proteins at the primed-FCSCs. The cell morphology and size did not differ in normal- and primed-FCSCs, and stem cell marker was maintained. Histologic analysis confirmed that the multilineage differentiation potential of FCSCs was maintained. CONCLUSIONS. In conclusion, this study demonstrated that CM of Primed-FCSCs showed higher ability to inhibit inflammatory events in synoviocytes than those of CM of unprimed-FCSCs. Moreover, probably upregulated anti-inflammatory factors mediated anti-inflammatory effects of FCSCs.
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Contents
Abstract--------------------------------------------------------------------------------------------ⅰ
Contents------------------ -------------------------------------------------------------------------ⅲ
List of Figures------------------------------------------------------------------------------------ⅳ
List of Table---------------------------------------------------------------------------------------ⅴ
1. Introduction--------------------------------------------------------------------------------------1
2. Materials and Methods-------------------------------------------------------------------------5
2.1 Isolation and Culture of Sem Cells-----------------------------------------------------5
2.2 Optimization of Priming Condition and Conditioned Media Preparation---------5
2.3 Anti-Inflammation Assay----------------------------------------------------------------6
2.4 RNA Analysis-----------------------------------------------------------------------------6
2.5 PGE2 Quantification----------------------------------------------------------------------6
2.6 Western Blot Analysis--------------------------------------------------------------------7
2.7 Human Cytokine Array------------------------------------------------------------------7
2.8 Cell Morphology Analysis---------------------------------------------------------------8
2.9 Flow Cytometry Analysis----------------------------------------------------------------8
2.10 Multilineage Differentiation-------------------------------------------------------------8
2.11 Statistical Analysis------------------------------------------------------------------------9
3. Results------------------------------------------------------------------------------------------11
3.1 Optimization of priming condition----------------------------------------------------11
3.2 FCSCs primed with TNF-α (10 ng/mL) and IL-1β (15 ng/mL) can down-regulate the level of pro-inflammatory markers in inflammatory synoviocytes----------15
3.3 Determination of protein mediating the anti-inflammatory effects of FCSCs--18
3.4 The stem cell character of FCSCs is maintained after priming-------------------21
4. Discussion-------------------------------------------------------------------------------------25
References------------------------------------------------------------------------------------------28
국문요약-------------------------------------------------------------------------------------------35
