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標題: | SIRT6可經由調控人類成骨細胞中失衡的葡萄糖代謝來減緩關節炎的進展 SIRT6 Attenuates Hypoxia-Induced Cyr61 Expression via Modulating Unbalanced Glycolysis in Human Osteoblastic Cells: A Therapeutic Potential for Arthritis |
作者: | Ling-Hsiu Chao 趙怜琇 |
指導教授: | 林思洸(Sze-Kwan Lin) |
關鍵字: | 類風濕性關節炎,缺氧,葡萄糖代謝,糖解作用,Cyr61,SIRT6, rheumatoid arthritis,hypoxia,glucose metabolism,glycolysis,Cyr61,SIRT6, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 類風濕性關節炎(rheumatoid arthritis, RA)是一種慢性的全身性免疫疾病,持續發炎造成RA關節長期處於一個缺氧(hypoxia)的狀態,過去文獻證實RA synovial tissue確實具有高度的糖解作用活性,過多的糖解作用所導致的代謝失衡,除了會刺激細胞造成不正常增生,糖解作用產物─乳酸的過度堆積也會導致細胞毒性,使得關節發炎的情形更加惡化。所以,如何改善這樣失衡的葡萄糖代謝情形,是一個值得探討的主題。
SIRT6為Sirtuins七個成員之一,目前已知和維持基因組的穩定性、防止老化、代謝以及發炎反應有關,尤其在維持生物體糖類代謝方面具有重要性。目前關於SIRT6能否調控類風溼性關節炎中失衡的糖類代謝,甚至藉此減緩發炎反應,都還是未知,這也是本研究所欲探討的主要目標。 在本次研究中,我們首先收集並分析不同病程進展的RA患者關節組織切片,初步證實SIRT6的表現與發炎程度具有臨床相關性。接著透過西方墨點法(Western blot)及乳酸含量檢測套組(Lactate assay kit)分析,我們發現SIRT6會減緩人類成骨細胞在缺氧環境下失衡的糖解作用,包含抑制乳酸脫氫脢(LDH; Lactate dehydrogenase)及降低乳酸 (Lactate)產量。我們更進一步發現SIRT6可以藉此機制去抑制Cyr61(cysteine rich protein 61) ,一個在RA患者的滑膜組織、FLS和關節液中均高度表達的蛋白質的表現。 為了更深入了解這之間的詳細機轉,根據本實驗室過去的實驗結果─Cyr61表現會受到轉錄因子CREB的調控,透過螢光素酶檢測法(Luciferase assay)及染色質免疫沉澱法(Chromatin Immunoprecipitation) 分析,我們發現在缺氧刺激下SIRT6會透過降低CREB結合到Cyr61啟動子的親和力去抑制Cyr61啟動子的活性。我們更進一步證明,乳酸會促進CREB對Cyr61啟動子的親和力,而SIRT6會透過抑制乳酸去抑制此一作用。 最後,我們透過動物實驗模式印證了在細胞實驗中所得到的結果。在膠原誘導關節炎大鼠模型中,我們發現SIRT6能抑制成骨細胞中 LDH及Cyr61的表現,並且能降低巨噬細胞的浸潤,具有抑制發炎進展、減緩骨吸收的作用。 總結本研究實驗結果,SIRT6可經由調控缺氧刺激下成骨細胞中失衡的葡萄糖代謝來降低Cyr61表現,藉此減緩關節炎的病程進展。期許此機制能夠成為RA治療上一個有潛力的新途徑。 Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by persistent synovitis. Evidence indicates that RA synovial tissues have increased glycolytic activity, which is related to the nature of hypoxia in RA synovium. Elevated production of lactate caused by unbalanced glycolysis stimulates abnormal cell proliferation and leads to joint destruction. Therefore, how to improve the situation of glucose metabolism is a topic worth exploring. Within the seven members of the sirtuin family, SIRT6 (sirtuin 6) is particularly involved in maintaining genomic stability, preventing age-related disorders, regulating metabolic processes and attenuating inflammation. It is unclear whether SIRT6 can play a role in regulating unbalanced glucose metabolism in RA. In this study, we demonstrated that SIRT6 suppressed Cyr61 expression via modulating increased glycolysis stimulated by hypoxia in osteoblastic cells. We also found that SIRT6 inhibited hypoxia-induced CREB phosphorylation and CREB-DNA binding. Hypoxia-induced Cyr61 promoter activation was dependent on CRE-CREB interaction and is inhibited by SIRT6. In rat collagen-induced arthritis (CIA), marked expression of Cyr61, LDH, CD68 was noted in the severe joint destruction group. In contrast, obvious SIRT6 was found in the group of mild severity. In conclusion, we have demonstrated that SIRT6 attenuates hypoxia-induced Cyr61 expression via modulating unbalanced glycolysis in osteoblastic cells and suppresses disease progression in rat CIA model. This finding may provide a new perspective on the treatment of RA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58966 |
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