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TNF-like weak inducer of apoptosis(TWEAK) induces inflammatory effect in Graves' orbital fibroblast

Other Titles
 갑상선안병증환자의 안와섬유모세포의 염증 병리기전에서 TNF-like weak inducer of apoptosis(TWEAK)의 역할 
Authors
 이성준 
College
 College of Medicine (의과대학) 
Degree
박사
Issue Date
2019
Abstract
Purpose: To investigate the role of effect of TNF-like weak inducer of apoptosis (TWEAK) on inflammatory pathogenesis in orbital fibroblasts in Graves’ orbitopathy (GO). Methods: Orbital fibroblasts were cultured from orbital adipose/connective tissues of patients with GO and healthy control subjects. Effects of treatment IL-1β and TNF-on TWEAK and its receptors TNFR-1, TNFR-2 and fibroblast growth factor-inducible 14(Fn14) messenger RNA (mRNA) and protein expressions were evaluated by real-time polymerase chain reaction (PCR) and Western blotting. To evaluate the role of TWEAK in inflammation, cells were first treated with exogenous TWEAK with various time (10 min -72h) and dose (10 – 500 ng/ml) to find mRNA and protein expression of pro-inflammatory cytokines including IL-6, IL-8, MCP-1. Other multiple cytokine mRNA expressions were investigated after treatment of rTWEAK. To find the role of Fn14 in the pathogenesis of TWEAK induced cytokine upregulation, Fn14 inhibitor (ITEM4) were treated prior to stimulation of cells with rTWEAK (100 ng/ml, 24h) and cytokines such as IL-6, IL-8 and MCP-1 mRNA and protein expression were analyzed using real time RT-PCR and ELISA. ELISA of hyaluronan was performed after treatment of rTWEAK with or without Fn14 inhibitor and other signal pathway inhibitors. Lastly, serum TWEK levels were measured and compared between 40 GO patients and 30 non-GO controls. Results: Expression levels of TWEAK and TNF- mRNA levels were significantly higher in GO tissue samples (n=11) than in non-GO tissue samples (n=7). TWEAK, TNFR-1, TNFR-2 and Fn14 mRNA, and TWEAK, Fn14 protein in GO orbital fibroblasts increased upon IL-1β and TNF- treatment. Treatment with rTWEAK significantly upregulated expression of pro-inflammatory cytokine mRNA including IL-6, IL-8, MCP-1. rTWEAK also significantly elevated protein secretion of IL-6, IL-8 and MCP-1 dose and time dependently, further in GO cells than in non-GO cells. Fn14 blocker, ITEM-4 as well as inhibitors of signal pathway molecules suppressed rTWEAK induced pro-inflammatory cytokine protein release. rTWEAK also elevated hyaluronan production, which were significantly suppressed by Fn14 blocker. Serum levels of TWEAK were significantly higher in GO patients (mean 328.7  67.3 pg/ml) than in non-GO control subjects (mean 231.0  43.6 pg/ml). Conclusion: Tissue and serum level of TWEAK was significantly higher in GO than in non-GO subjects. IL-1β and TNF- upregulated TWEAK and receptor expression and vice versa, rTWEAK also stimulated production of pro-inflammatory cytokines more in GO cells. Blocking Fn14, TWEAK receptor led to decreased production of pro-inflammatory cytokines and hyaluronan which were reported main in the pathogenesis of GO. Thus, modulation of TWEAK activity might have therapeutic potential in the suppression of inflammation and remodeling in GO. 목적: 갑상선안병증의 안와섬유모세포에서 TNF-like weak inducer of apoptosis (TWEAK) 이 염증 병리기전에 미치는 영향을 알아보고자 하였다. 방법: 갑상선안병증 환자 및 건강한 대조군의 안와 지방조직으로부터 안와 섬유모세포를 배양하였다. TWEAK, TNFR-1, TNFR-2와 Fn14 mRNA의 발현정도가 갑상선안병증과 건강한 대조군 안와조직간, 그리고 혈청에서 차이가 있는지를 실시간 중합 효소 연쇄 반응 및 ELISA를 이용하여 비교평가 하였다. 염증에 대한 TWEAK의 역할을 평가하기 위해, 일차배양한 안와섬유모세포를 최소 3개의 샘플이상, IL-1b와 TNF-a 자극을 주고 TWEAK, Fn14 mRNA와 단백질 발현정도를 평가하였고, 반대로 recombinant TWEAK을 외부에서 처리한 후, 안와섬유모세포에서 IL-6, IL-8, MCP-1과 같은 염증성 사이토카인의 mRNA, 단백질 발현 정도를, 갑상선안병증과 정상 안와섬유모세포에서 비교 평가하였다. Fn14 항체를 전처치 하여, TWEAK에 의해 증가된 사이토카인이 억제되는지를 ELISA를 이용하여 분석하였고, TWEAK에 의해 하이루론산이 증가한 것을 확인한 후, 이것이 Fn14 항체에 의해 억제되는지를 ELISA를 이용하여 평가하였다. 결과: TWEAK의 mRNA 발현이 갑상선안병증 안와조직에서 더 발현이 증가되어 있었으며, 갑상선안병증 환자 혈청에서 TWEAK농도가 정상인보다 더 증가해 있었다. IL-1와 TNF-는 갑상선안병증 안와섬유모세포에서 TWEAK mRNA와 단백질 농도를 더 증가시켰으며, recombinant TWEAK자극을 한 경우 IL-6, IL-8, MCP-1 mRNA와 단백질이 용량 비례적으로, 갑상선안병증 안와섬유모세포에서 더 증가하였다. Fn14항체를 이용하여 Fn14를 억제하면, TWEAK에 의해 증가한 IL-6, IL-8, MCP-1 단백질 농도가 의미있게 감소되었으며, TWEAK에 의해 증가한 히아루론산이 의미있게 억제되는 것이 관찰되었다. 또한, 갑상선안병증 환자의 혈청 TWEAK이 (40명, 328.7  67.3 pg/ml) 정상인 혈청 TWEAK (30명, 231.0  43.6 pg/ml) 보다 의미 있게 높았다. 결론: 갑상선안병증 환자에서 TWEAK의 biomarker로서 역할을 기대해 볼 수 있으며, 갑상선안병증 환자의 안와조직에서 일차배양한 안와섬유모세포에서 TWEAK은 염증 기전과 밀접한 관련이 있을 것으로 생각된다. Fn14의 차단이 염증 관련 사이토카인의 생성과 리모델링 관련 히아루론산 생성을 억제시킨 것은, TWEAK/Fn14을 조절함으로써 fff..갑상선안병증 초기 염증 단계에서 치료적 효과를 기대해볼 수 있겠다.
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1. College of Medicine (의과대학) > Others (기타) > 5. Others
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/178210
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