臺灣博碩士論文加值系統

退化性關節炎是成人中最常見的關節炎疾病，主要的臨床症狀是關節疼痛、變形、活動度受限。其X光上的特徵包括由於軟骨的磨損、基底骨質硬化，而造成關節腔狹窄及骨刺的增生。目前已知形成退化性關節炎的危險因子包括老化、關節受傷或過度使用、體重過重等，然而對於其致病機制仍不甚了解，本論文主要目的為探討人類軟骨細胞受巨噬細胞及剪力作用下，urokinase plasminogen activator (uPA)基因表現之分子機制，以及透過對老鼠成骨細胞增生、礦物質化以及蝕骨細胞分化的影響，來評估薑黃素奈米化微脂體對於退化性關節炎之保護效果。
將人類周邊血液巨噬細胞培養後，收集其條件培養液(conditioned medium)。人類軟骨細胞受到巨噬細胞條件培養液刺激後，透過細胞內訊息傳遞途徑JNK/Akt的磷酸化及轉錄因子NF-κB活化，誘導uPA基因表現。利用IL-1ra抑制劑進一步發現條件培養液中之IL-1對於uPA之誘導扮演重要角色。實驗證據顯示低程度剪力作用能經由單磷酸腺苷活化蛋白激酶(AMPK)的活化，有效抑制巨噬細胞條件培養液誘導之軟骨細胞JNK/Akt的磷酸化及轉錄因子NF-κB的活化，並抑制uPA基因表現增加。因而進一步研究單磷酸腺苷活化蛋白激酶對軟骨細胞的保護機轉, 可能對退化性關節炎的治療開一扇門。
此外，薑黃素(curcumin，Cur)與二去甲基薑黃素(bisdemethoxy- curcumin，BDMC) 是由薑黃(Curcuma longa) 所萃取出來的非水溶性多酚類抗發炎化合物，在過去的研究指出具有良好的抗發炎效果，可以應用在退化性關節炎的治療上。為改善薑黃素與二去甲基薑黃素被細胞利用的方式，大豆磷脂質(soybean phosphatidylcholine)用來包覆薑黃素或二去甲基薑黃素製成奈米化微脂體(Cur-Lip和BDMC-Lip)。結果顯示微脂體對Cur 和 BDMC的包覆率約為70%，並且其大小穩定並可被細胞吸收。Cur-Lip和BDMC-Lip兩者皆可抑制巨噬細胞受刺激後發炎因子一氧化氮(nitric oxide；NO)的產生與朝蝕骨細胞的分化。此外與Cur 和 BDMC比較，Cur-Lip和BDMC-Lip的細胞毒性顯著下降。與BDMC-Lip相比, Cur-Lip更可以維持成骨細胞的增生及礦物質化功能。在介白素(Interleukin (IL)-1β)的刺激下，Cur-Lip和BDMC-Lip可以降低成骨細胞的發炎因子的表現並增加成骨細胞抑制分子(Osteoprotegerin；OPG)對NF-κB配位體接受器活化因子(receptor activator for NF-κB ligand；RANKL)的比值，有效減緩骨質的代謝。因此我們研究發現微脂體包覆薑黃素與二去甲基薑黃素兩種藥物可以有效地降低蝕骨細胞活化和維持成骨細胞的功能，達到保護效果延緩退化性關節炎的進程。

Osteoarthritis (OA) is the most common degenerative joint disease in the world. The clinical symptoms are pain, stiffness and deformation of affected joints which are characterized with progressive destruction of articular cartilage, joint space narrowing, subchondral sclerosis and spur formation. Aging, joint trauma, obesity and excessive repetitive loading are the well-known risk factors of OA, but the exact mechanisms of OA remain unclear. The aim of this thesis was to investigate the mechanisms underlying macrophage-induced urokinase plasminogen activator (uPA) expression in human chondrocytes and its modulation by fluid shear stress; and to evaluate the protective effects of curcuminoid-loaded liposomes against bone turnover in a cell-based model of osteoarthritis. This thesis addresses the preparation of peripheral blood-macrophage-conditioned medium (PB-MCM), modeling of shear stress in a parallel plate flow chamber, liposome formulation techniques, cell uptake and bioactivity assays.
Stimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that the JNK/Akt phosphory- lation and NF-κB activation are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK/Akt phosphorylation, NF-κB activation, and uPA expression. The PB- MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression. Therefore, our data support the hypothesis that uPA upregulation stimulated by macrophages may be modurated with low fluid shear stress. AMPK may potentially provide new molecular target or new ‘entry point’ to develop an effective disease-modifying drugs for the treatment of osteoarthritis.
In addition, curcumin (Cur) and bisdemethoxycurcumin (BDMC), extracted from Curcuma longa, are poorly-water-soluble polyphenol compounds that have shown antiinflammatory potential for the treatment of osteoarthritis. To increase cellular uptake of Cur and BDMC in 7F2 mouse osteoblastic cells, soybean phosphatidylcholine (SPC) were used for liposome formulation. There are about 70% entrapment efficiency of Cur and BDMC in the liposomes and the particle sizes are stable after liposome formation. Both Curcuminoid-loaded liposomes can inhibit NO production of macrophage and its osteoclast differential activities. In comparison with free drugs (Cur and BDMC), curcuminoid-loaded liposomes were less cytotoxic, and expressed high cellular uptake of the drugs. Of note is that Cur-Lip can prevent liposome-dependent inhibition of osteoblast differentiation and mineralization, but BDMC-Lip could not. With interleukin (IL)-1β stimulation, curcuminoid-loaded liposomes can successfully down- regulate the expression of inflammatory markers on osteoblasts, and show high osteoprotegerin (OPG)/ Receptor activator of nuclear factor κB ligand (RANKL) ratio to prevent osteoclastogenesis. Our data demonstrated that Cur and BDMC can be successfully encapsulated in liposomes and that can reduce osteoclast activity and maintain osteoblast functions. Therefore, Curcuminoid-loaded liposomes may slow osteoarthritis progression.

目 錄
指導教授推薦書
口試委員會審定書
誌謝……………………………………………………………………...iii
中文摘要………………………………………….……………………..iv
英文摘要……………………………………………………………. .....vi
目錄……………………………………………………………......……..viii
Directory of Figures……………………………...……………..………...xii
Directory of Tables …………………………………….....……..…....…..xiv
Chapter 1 Introduction……………………………………………..1
1.1 Overview of osteoarthritis .. ………….…………………………2
1.2 Pathogenesis of osteoarthritis…..………………………………..4
1.3 Structure of articular cartilage …………………………………..6
1.4 Interaction of Growth factors and proinflammatory Cytokines.…8
1.5 MMP/ ADAMTS/ TIMMP , UPA/ plasmin/ PAI-I…….………11
1.6 Mechanobiology of cartilage………..………………..………….13
1.7 Diverse response to fluid Shear stress on osteoarthritic lesional and non-lesional chondrocytes……………………………...…..16
Chapter 2 Moduration of macrophage-induced urokinase plasminogen activator expression in human chondrocytes under fluid shear stress…………21
2.1 Purpose………………………………………………………….22
2.2 Materials and methods………………………………………….23
2.2.1 Reagents……………………………………………………....23
2.2.2 Culture of human chondrocytes………………………………24
2.2.3 Isolation of peripheral blood monocytes……………………...24
2.2.4 Preparation of peripheral blood monocyte-derived macrophageconditioned medium (PB-MCM)……………………………...25
2.2.5 Shear-stress experiment………………………………………25
2.2.6 Real-time quantitative PCR…………………………………..26
2.2.7 Quantification of uPA expression…………………………….26
2.2.8 Western blot analysis…………………………………………26
2.2.9 Reporter gene constructs, siRNA, transfection, and luciferase assays………………………………………………………….27
2.2.10 Chromatin immunoprecipitation assay (ChIP)……………...27
2.2.11 Transcription factor ELISA assay…………………………...28
2.2.12 Statistical analysis…………………………………………...28
2.3 Results…………………………………………………………..28
2.3.1 Conditioned medium from macrophages induces the upregulation of uPA in human chondrocytes………………...28
2.3.2 PB-MCM-induced uPA expression is mediated by the JNK and Akt signaling pathways…………………………29
2.3.4 NF-κB binding sites are major determinants of the PB-MCM induction of uPA promoter activity………………...30
2.3.5 The JNK and Akt signaling pathways are involved in macrophage- induced uPA promoter activity………………31
2.3.6 IL-1ra inhibits macrophage-induced signaling transduction and uPA expression…………………………....………32
2.3.7 Exposure of human chondrocytes to shear stress of 2 and 5 dyn/cm2 inhibits macrophage-induced uPA expression……33
2.3.8 Effect of AMPK on PB-MCM-induced uPA expression….….33
2.4 Conclusion……………………………………………………34
Chapter 3 Evaluation of the protective effects of curcuminoid- loaded liposomes (Cur-Lip and BDMC-Lip) against bone turnover in a cell-based model of osteoarthritis………………………..35
3.1 Purpose……………………………………………….………….36
3.2 Materials and methods…………………………………………...38
3.2.1 Cell culture and Reagents……………………………………...38
3.2.2 Liposome formulations………………………………………...39
3.2.3 Particle characterization………………………………………..39
3.2.4 Cell viability and Proliferation assay…………………………..40
3.2.5 Entrapment efficiency………………………………………….40
3.2.6 Fluorescence spectroscopy method for intracellular uptake of liposomes………………………………41
3.2.7 Nitrite measurement…………………………………………...42
3.2.8 Osteoclast differentiation assay………………………………..42
3.2.9 Cellular alkaline phosphatase (ALP) activity………………….43
3.2.10 Mineralization of the extracellular matrix…………………....44
3.2.11 Quantitative real-time PCR……………………….…………..44
3.2.12 Statistical analysis…………………………………………….45
3.3 Results………………………………………………………......45
3.3.1 In Vitro Characterization of liposomes……... …………………45
3.3.2 Effect of liposomal formulations on the cell viability, delivery efficiency and NO production…………………………………47
3.3.3 Inhibitory effects of Curcuminoid-loaded liposomes on osteoclast differentiation in RAW264.7 macrophages…………48
3.3.4 Effect of 7F2 osteoblasts by curcuminoid-loaded liposome.…...........49
3.3.5 Curcuminoid-loaded liposome protective osteoblast when stimulated with IL-1β…………………………………………51
3.4 Conclusion……………………………………….…………….52
Chapter 4 Discussion ……………………………………………54
References………………………………………………………………94


Directory of Figures
Fig 1. Induction of urokinase-type PA (uPA) in chondrocytes by peripheral blood-macrophage-conditioned medium (PB-MCM) stimulation.................................68
Fig 2. The JNK and Akt pathways are required for peripheral blood- macrophage-conditioned medium (PB-MCM)-induced urokinase- type PA (uPA) expression............69
Fig 3. The role of nuclear factor-κβ (NF-κβ) in peripheral blood- macrophage-conditioned medium (PB-MCM)-induced urokinase- type PA (uPA) mRNA expression and promoter activity..................................................................71
Fig 4. The JNK and Akt signaling pathways are involved in peripheral blood-macrophage-conditioned medium (PB-MCM)-induced urokinase- type PA (uPA) promoter activity...........................................................................73
Fig 5. Interleukin (IL)-1β is the major factor underlying peripheral blood-macrophage-conditioned medium (PB-MCM)-induced urokinase- type PA (uPA) expression in chondrocytes.......................................................................75
Fig 6. Effects of interleukin (IL)-1ra on peripheral blood- macrophage- conditioned medium (PB-MCM)-induced signaling in chondrocytes..................................76
Fig 7. Exposure of chondrocytes to lower shear stress inhibits peripheral blood-macrophage-conditioned medium (PB-MCM)-induced urokinase-type PA (uPA) expression.....77
Fig 8. Effect of AMPK on peripheral blood-macrophage-conditioned medium (PB-MCM)-induced urokinase-type PA (uPA) expression ..................................79
Fig 9. Particle size of liposome formulations......................................80
Fig 10. The effect of Curcuminoid-loaded liposomes on cell viability...............81
Fig 11. Cell uptake of liposomes on 7F2 osteoblastic cells.........................82
Fig 12. Effects of curcuminoid-loaded liposomes on nitric oxide (NO) production in LPS (500ng/ml) induced RAW264.7 macrophages for 24 hours...........................83
Fig 13. curcuminoid-loaded liposomes (70μM) suppress osteoclastogenesis in 10ng/ml LPS and 50ng/ml RANKL induced RAW264.7 macrophages after 5 days incultivation....….84
Fig 14. Effects of curcuminoid-loaded liposomes (70μM) on osteoblast proliferation, differentiation and mineralization.................................................86
Fig 15. Effects of curcuminoid-loaded liposomes (70μM) on the expression of pro- inflammatory mediators (cyclooxygenase 2; COX-2), metalloproteinase-3 (MMP-3), and OPG/RANKL ratio of 7F2 osteoblastic cells in the presence or absence of 10ng/ml IL-1β stimulation........................................................................89
Fig16. curcuminoid-loaded liposomes (70 µM) induced adipogenesis in 7F2 osteoblastic cells after 7 days of cultivation..................................................91


Directory of Tables
Table 1. Sequences of primers used in real-time PCR experiment.....................92
Table 2. Encapsulation parameters of the liposomal formulations....................93

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