臺灣博碩士論文加值系統

低氧誘導的醣解作用及氧化還原訊息傳遞在發炎性骨吸收的相關機制還不清楚。過往的文獻指出SIRT6在葡萄糖代謝及氧化還原訊息傳遞扮演重要的腳色。本篇研究中我們探討在人類成骨細胞(human osteoblast)中醣解作用和過氧化物(reactive oxygen species，ROS)生成之間的關聯性，並研究SIRT6是否會透過調節這些途徑來影響發炎反應。人類成骨細胞在低氧環境下，我們測量乳酸脫氫酶(lactate dehydrogenase A, LDHA)、乳酸及過氧化物的產生，並探討乳酸及過氧化物之間的相互關係，了解這兩個機制對於誘導發炎因子的影響。透過成骨細胞過量表現SIRT6的方式了解SIRT6是如何影響過氧化物生成及糖解作用。在大鼠關節炎模式中，我們利用lentivirus基因治療的方式將SIRT6基因或empty vector表現在關節中，利用免疫組織染色測量成骨細胞LDHA、Cyr61及巨噬細胞的吸引在發炎程度中的相關性。我們的結果顯示低氧會促進LDHA蛋白的表現和過氧化物的生成，兩者之間利用正回饋的方式相互活化。另外低氧會促進發炎因子Cyr61、TNF-α、IL-1β和IL-6的表現，並且這個現象會透過SIRT6調控醣解作用和過氧化物的生成所抑制。在關節炎動物模式中，利用基因治療的方式將SIRT6過量表現於關節時，可以減緩發炎反應、骨細胞合成Cyr61及巨噬細胞的吸引，同時也會降低LDHA及氧化性病灶組織的量。我們的結果顯示出SIRT6可藉由調控葡萄糖代謝及氧化還原平衡來減緩成骨細胞的發炎反應。在未來藉由調控代謝重組的機制，可能成為治療發炎性骨吸收的一個治療策略。

Relation between hypoxia-enhanced glycolysis and redox signaling in inflammatory bone diseases is unclear. Sirtuin 6 (SIRT6) is pivotal for glucose metabolism and redox homeostasis. In the study we examined the connection between glycolysis and reactive oxygen species (ROS) production in human osteoblasts (HOB) and assessed whether SIRT6 modulates inflammation via regulation of these activities. In HOB cultured under hypoxia, expression of lactate dehydrogenase A (LDHA), lactate production and ROS generation were examined. The reciprocal effects between lactate and ROS production and their impact on inflammatory cytokine induction were assessed. The action of SIRT6 on the above reactions was determined. In a rat model of collagen-induced arthritis (CIA), the relation between inflammatory activity and osteoblastic expression of LDHA, oxidative lesions, Cyr61 synthesis and macrophage recruitment were evaluated in joints with or without lentiviral-SIRT6 gene therapy. Results showed that hypoxia enhanced lactate and LDHA production in HOB. ROS generation also increased and there was a positive feedback between glycolysis and ROS formation. Overexpression of SIRT6 attenuated hypoxia-enhanced glycolysis and ROS generation. Hypoxia-induced expressions of Cyr61, TNF-α, IL-1β and IL-6 were suppressed by SIRT6 and the inhibitory effects overlapped with anti-glycolytic and anti-oxidation mechanisms. In CIA, overexpression of SIRT6 ameliorated inflammation, osteoblastic synthesis of Cyr61 and macrophage recruitment. Expression of LDHA and oxidative lesions were decreased in osteoblasts of SIRT6-treated joints. Our findings suggest that SIRT6 suppresses inflammatory response in osteoblasts via modulation of glucose metabolism and redox homeostasis. Reprogramming cellular metabolism may possess therapeutic potential for inflammatory bone resorption.

口試委員審定書 1
中文摘要 2
Abstract 3
第一章 導論 5
1.1 低氧與類風溼性關節炎 5
1.2 低氧對粒線體的影響 6
1.3 乳酸對類風溼性關節炎的影響 8
1.4 ROS對細胞的影響 9
1.5 SIRT6與發炎反應的關聯 10
1.6 趨化因子CCL2與monocyte的浸潤 11
第二章 材料與方法 12
2.1 抗體列表 12
2.2 人類骨髓成骨細胞初待培養 12
2.3 總量ROS測量 12
2.4 西方點墨法 13
2.5 乳酸測量 13
2.6 HOB細胞利用慢病毒SIRT6基因感染過度表現及shRNA抑制 14
2.7 定量型即時聚合酶鏈式反應 (qRT-PCR) 14
2.8 關節炎動物模式 15
2.9 動物關節炎臨床上的診斷 16
2.10 動物關節炎影響上的評量 16
2.11 免疫組織染色及組織學分析 17
2.12 統計分析 17
第三章 結果 18
3.1 低氧會誘導人類成骨細胞ROS的生成及促進醣解作用 18
3.2 醣解作用和ROS生成的正回饋現象 18
3.3 SIRT6會抑制低氧所誘導的ROS生成及醣解作用 19
3.4 SIRT6可藉由抑制ROS及醣解作用降低低氧所促進的發炎因子合成 19
3.5 SIRT6在關節炎動物模式上的治療潛力 20
3.6 SIRT6抑制發炎關節中成骨細胞的ROS生成及醣解作用 21
第四章 討論 22
4.1 SIRT6在類風溼性關節炎扮演的角色 22
4.2 SIRT6調控醣解作用和氧化壓力的機制 23
4.3 SIRT6在關節炎動物模式中扮演的角色 24
4.4 總結 25
4.5 未來展望 25
參考資料 26
圖一 低氧促進成骨細胞ROS的生成 39
圖二 低氧促進LDHA蛋白表現 40
圖三 低氧促進乳酸的分泌 41
圖四 ROS促進醣解作用 42
圖五 乳酸促進ROS生成 43
圖六 SIRT6抑制低氧所促進的ROS生成 44
圖七 SIRT6抑制低氧所促進的醣解作用 45
圖八 SIRT6抑制低氧所促進的醣解作用 46
圖九 成骨細胞藉由轉染LDHA shRNA抑制表現 47
圖十 SIRT6藉由抑制ROS/醣解作用降低Cyr61的表現 48
圖十一 SIRT6藉由抑制ROS/醣解作用降低TNF-a的表現 49
圖十二 SIRT6藉由抑制ROS/醣解作用降低IL-1b的表現 50
圖十三 SIRT6藉由抑制ROS/醣解作用降低IL-6的表現 51
圖十四 IVIS imaging system分析SIRT6基因治療的表現 52
圖十五 SIRT6基因治療可減緩關節炎腫脹程度 53
圖十六 Arthritis Index 54
圖十七 Radiographic Score 55
圖十八 empty vector HE染色 56
圖十九 SIRT6基因治療HE染色 57
圖二十 empty vector SIRT6 IHC染色 58
圖二十一 SIRT6基因治療組SIRT6 IHC染色 59
圖二十二 empty vector LDHA IHC染色 60
圖二十三 empty vector 8-OHdG IHC染色 61
圖二十四 empty vector Cyr61 IHC染色 62
圖二十五 empty vector CD68 IHC染色 63
圖二十六 SIRT6基因治療組LDHA IHC染色 64
圖二十七 SIRT6基因治療組8-OHdG IHC染色 65
圖二十八 SIRT6基因治療組Cyr61 IHC染色 66
圖二十九 SIRT6基因治療組CD68 IHC染色 67
表一 Semi-quantitative analyses of histopathology and immunohistochemistry of CIA joints injected with empty pLVX vector or pLVX-SIRT6. 68
附錄一 歷年文獻發表 69
附錄二 畢業論文文稿一 71
附錄三 畢業論文文稿二 82

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