臺灣博碩士論文加值系統

類風濕性關節炎 (rheumatoid arthritis，簡稱RA) 是一種自體免疫功能失調造成之慢性發炎疾病，可導致關節發炎甚至失去功能。特徵包括免疫細胞浸潤於關節腔、滑膜細胞活化及增生、血管新生、發炎性趨化激素及細胞激素的釋放，進而引起分解性酵素分泌導致軟、硬骨受到破壞。類鐸受體 (toll like receptor，簡稱TLR) 為一群可透過辨識病原相關分子型態 (pathogen associated molecular patterns，簡稱PAMPs) 而誘導核轉錄因子κB (nuclear factor kappa B，簡稱NF-κB)及干擾素調控因子 (interferon-regulatory factor，簡稱IRF) 訊息傳遞的膜蛋白， 過去文獻指出可辨識單股核醣核酸 (single strandribonucleic acid，簡稱ssRNA) 的類鐸受體七號 (TLR7) 在RA 病人之關節組織有高量表現的情況。因此在本實驗中，我們首先偵測在膠原蛋白誘發之大鼠關節炎 (collagen-induced arthritis，簡稱CIA)發病過程中TLR7 表現，發現在關節炎發病初期至中期，大鼠關節萃取液中TLR7表現量較正常大鼠表現量高，因此我們進一步構築帶有TLR7核酸干擾因子 (RNA interference，簡稱RNAi) 之慢病毒，命名為Lt.shTLR7，進一步評估Lt.shTLR7 在動物體及細胞實驗中抑制TLR7的效果。我們發現在Lt.shTLR7 治療之CIA 大鼠，其關節炎之臨床症狀、X 光影像 (radiography) 與組織病理學 (histology) 上之病理特徵都有改善的情況。此外，Lt.shTLR7 治療之大鼠可減緩血管新生、T細胞浸潤數目及細胞激素介白素六號 (interleukin-6，簡稱IL-6) 的釋
放。我們進一步利用TLR7 受質imiquimod (INN) 刺激滑膜細胞後發
現只有滑膜纖維母細胞 (synovial fibroblast，簡稱SF) 可隨INN 的劑量增高而促使血管內皮生長因子 (vascular endothelial growth factor，簡稱VEGF) 釋放情況增加，且參與RA 血管新生的過程。最後我們也發現CIA 關節滑液中刺激SF TLR7 訊息可受到核醣核酸酶及內質網成熟抑制劑的抑制，這也意味著關節滑液中確實存在TLR7 的內生性RNA 受質。總結來說，本實驗旨在對RA 的致病及臨床應用研究上提供一個不同於以往的觀點，利用慢病毒載體攜帶TLR7 的RNA干擾因子提供長效性的基因沉默作用以達到治療關節炎的目的。

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that causes loss of function in the joints. It is characterized by infiltration of immune cells into the joints, activation of synovial cells, and production of inflammatory cytokines together with degradative enzymes that progressively leads to the cartilage and bone destruction. Toll like receptors (TLRs) are a family of membrane proteins that trigger the nuclear factor-κB-dependent signaling pathway. Recent studies have shown that TLR7, the receptor of single strand RNA (ssRNA), is highly expressed in the RA joints. In this study, we firstly demonstrated that TLR7 expression was unregulated in the ankle joint tissue extracts during disease progression in a rat model of collagen-induced arthritis (Firestein et al.). Furthermore, we constructed short-hairpin RNA of rat TLR7 gene to a lentivirial vector, designated Lt.shTLR7, and evaluated the efficiency of Lt.shTLR7 to knock-down TLR7 gene expression both in vitro and in vivo. Intra-articular administration of Lt.shTLR7 reduced articular circumference and index, radiological score, and histological score in rats with CIA. Moreover, Lt.shTLR7-treated rats showed reduced angiogenesis and T cells infiltration. We further used TLR7 ligand imiquimod to stimulate synoviocytes and found that imiquimod could induce synovial fibroblsts (SFs) release vascular endothelial growth factor (VEGF), that is involved in the process of angiogenesis in the pathogenesis of RA. Finally, we demonstrated that pretreatment of CIA rat joint fluid with RNase and chloroquine could abolish TLR7 signaling in SFs, suggesting that ssRNA ligand was present in arthritic joint fluid. Taken together, our studies provided different insights into the pathogenesis and potential therapy of RA by utilization of lentiviral vector expressing TLR7 shRNA for the long-term gene silencing.

中文摘要................................................1
Abstract ...............................................3
致謝....................................................5
目錄....................................................6
圖目錄..................................................8
縮寫與符號..............................................10
緒論....................................................11
A. 類風濕性關節炎 (Rheumatoid arthritis，簡稱RA) .......11
1. 特徵.................................................11
2. 致病機轉.............................................11
B. 類鐸受體 (toll like receptor，簡稱TLR) ..............13
1. 概論.................................................13
2. TLR 與RA ............................................14
3. TLR 與血管新生.......................................15
4. 類鐸受體7 號 (toll like receptor 7，簡稱TLR7)........15
C. 基因治療(gene therapy)...............................16
F. 研究動機與實驗設計...................................20
材料與方法..............................................21
一、 材料...............................................21
1. 質體(Plasmid) .......................................21
2. 寡聚核苷酸 (oligodeoxynucleotides，簡稱ODN)..........21
3. 細胞株 (cell line) ..................................23
4. 動物.................................................24
二、 方法...............................................24
1. 細胞株與細胞培養.....................................24
2. 西方墨點法 (western blot analysis) ..................25
3. 慢病毒載體之篩選及構築 (pLVTHM/shTLR7 construction) .26
4. 慢病毒生產 (Production of lentivirus vector).........28
5. 慢病毒效價偵測 (Virus titration).....................29
6. 反轉錄酶-聚合酶連鎖反應(Reverse transcriptase-polymerase chain reaction, RT-PCR)......................30
7. 第二型膠原蛋白誘導之大鼠關節炎模式 (typeⅡcollagen-induced arthritis，簡稱CIA) 及慢病毒之給予..............31
8. 大鼠關節炎臨床症狀之評估.............................31
9. X 光攝影評估.........................................32
10. 組織病理學評估及免疫組織染色 (Immunohistochemical analysis) ..............................................32
11. 酵素免疫分析法 (Enzyme-linked immunosorbent assay，簡稱ELISA)..................................................34
12. 統計分析 (Statistical analysis) ....................35
結果....................................................36
一、關節中CIA-SF 內生性TLR7 蛋白在疾病發生過程早期表現增加......................................................36
二、 關節滑液中存在內生性配體 (endogenous ligand) RNA 刺激CIA-SF 釋放細胞激素.....................................36
三、 TLR7 的liagnd INN 可促進CIA-SF 血管內皮生長因子 (VEGF)、介白素六號 (IL-6) 的釋放及干擾素誘導蛋白十號 (IP-10) 之表現..............................................37
四、 TLR7 shRNA 之構築與篩選............................39
五、 慢病毒載體pLVTHM/ shTLR7 之構築....................40
六、 慢病毒之生產及其抑制效果...........................40
七、 在膠原蛋白誘導之大鼠關節模式中，Lt.shTLR7 可有效預防性治療CIA大鼠關節炎之症狀及減少X 光照像 (radiographic) 與組織學(histologic) 上之病理特徵.............................42
八、 Lt.shTLR7 在CIA 大鼠滑膜中具有降低血管新生的功能...44
九、 Lt.shTLR7 在CIA 大鼠滑膜中具有降低T 細胞之浸潤及發炎反應細胞激素的功能........................................45
討論....................................................47
參考文獻................................................51
圖......................................................55
附圖....................................................69

Angelo, L. S., and Kurzrock, R. (2007). Vascular endothelial growth factor and its relationship to inflammatory mediators. Clin Cancer Res 13, 2825-2830.
Arancibia, S. A., Beltran, C. J., Aguirre, I. M., Silva, P., Peralta, A. L., Malinarich, F., and Hermoso, M. A. (2007). Toll-like receptors are key participants in innate immune responses. Biol Res 40, 97-112.
Berghofer, B., Frommer, T., Haley, G., Fink, L., Bein, G., and Hackstein, H. (2006). TLR7 ligands induce higher IFN-alpha production in females. J Immunol 177, 2088-2096.
Brenner, S., and Malech, H. L. (2003). Current developments in the design of onco-retrovirus and lentivirus vector systems for hematopoietic cell gene therapy. Biochim Biophys Acta 1640, 1-24.
Brentano, F., Kyburz, D., Schorr, O., Gay, R., and Gay, S. (2005a). The role of Toll-like receptor signalling in the pathogenesis of arthritis. Cell Immunol 233, 90-96.
Brentano, F., Schorr, O., Gay, R. E., Gay, S., and Kyburz, D. (2005b). RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3. Arthritis Rheum 52, 2656-2665.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.
Corr, M. (2005). The tolls of arthritis. Arthritis Rheum 52, 2233-2236.
Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., and Reis e Sousa, C. (2004). Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529-1531.
Ehlers, M., and Ravetch, J. V. (2007). Opposing effects of Toll-like receptor stimulation induce autoimmunity or tolerance. Trends Immunol 28, 74-79.
Firestein, F., Rozenszajn, L. A., Shemesh-Darvish, L., Elimelech, R., Radnay, J., and Rosenschein, U. (2003). Induction of apoptosis by ultrasound application in human malignant lymphoid cells: role of mitochondria-caspase pathway activation. Ann N Y Acad Sci 1010, 163-166.
Firestein, G. S., Alvaro-Gracia, J. M., and Maki, R. (1990). Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J Immunol 144, 3347-3353.
Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1, 27-31.
Frantz, S., Vincent, K. A., Feron, O., and Kelly, R. A. (2005). Innate immunity and angiogenesis. Circ Res 96, 15-26.
Gorden, K. B., Gorski, K. S., Gibson, S. J., Kedl, R. M., Kieper, W. C., Qiu, X., Tomai, M. A., Alkan, S. S., and Vasilakos, J. P. (2005). Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol 174, 1259-1268.
Gould, D. J., and Favorov, P. (2003). Vectors for the treatment of autoimmune disease. Gene Ther 10, 912-927.
Grimm, D., and Kay, M. A. (2007). Therapeutic application of RNAi: is mRNA targeting finally ready for prime time? J Clin Invest 117, 3633-3641.
Huber, L. C., Distler, O., Tarner, I., Gay, R. E., Gay, S., and Pap, T. (2006). Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology (Oxford) 45, 669-675.
Hurst, J., and von Landenberg, P. (2008). Toll-like receptors and autoimmunity. Autoimmun Rev 7, 204-208.
Jou, I. M., Shiau, A. L., Chen, S. Y., Wang, C. R., Shieh, D. B., Tsai, C. S., and Wu, C. L. (2005). Thrombospondin 1 as an effective gene therapeutic strategy in collagen-induced arthritis. Arthritis Rheum 52, 339-344.
Khoury, M., Jorgensen, C., and Apparailly, F. (2007). RNAi in arthritis: prospects of a future antisense therapy in inflammation. Curr Opin Mol Ther 9, 483-489.
Koch, A. E. (2003). Angiogenesis as a target in rheumatoid arthritis. Ann Rheum Dis 62 Suppl 2, ii60-67.
Krieg, A. M. (2007). The toll of too much TLR7. Immunity 27, 695-697.
Krieg, A. M., and Vollmer, J. (2007). Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunol Rev 220, 251-269.
Liu, Z. Q., Deng, G. M., Foster, S., and Tarkowski, A. (2001). Staphylococcal peptidoglycans induce arthritis. Arthritis Res 3, 375-380.
Marshak-Rothstein, A. (2006). Toll-like receptors in systemic autoimmune disease. Nat Rev Immunol 6, 823-835.
McIntyre, G. J., and Fanning, G. C. (2006). Design and cloning strategies for constructing shRNA expression vectors. BMC Biotechnol 6, 1.
Mocellin, S., and Provenzano, M. (2004). RNA interference: learning gene knock-down from cell physiology. J Transl Med 2, 39.
Muller-Ladner, U., Pap, T., Gay, R. E., Neidhart, M., and Gay, S. (2005). Mechanisms of disease: the molecular and cellular basis of joint destruction in rheumatoid arthritis. Nat Clin Pract Rheumatol 1, 102-110.
Naguwa, S. M. (2005). Tumor necrosis factor inhibitor therapy for rheumatoid arthritis. Ann N Y Acad Sci 1051, 709-715.
Nakajima, A. (2006). Application of cellular gene therapy for rheumatoid arthritis. Mod Rheumatol 16, 269-275.
O'Dell, J. R. (2004). Therapeutic strategies for rheumatoid arthritis. N Engl J Med 350, 2591-2602.
Ospelt, C., Kyburz, D., Pierer, M., Seibl, R., Kurowska, M., Distler, O., Neidhart, M., Muller-Ladner, U., Pap, T., Gay, R. E., and Gay, S. (2004). Toll-like receptors in rheumatoid arthritis joint destruction mediated by two distinct pathways. Ann Rheum Dis 63 Suppl 2, ii90-ii91.
Pinhal-Enfield, G., Ramanathan, M., Hasko, G., Vogel, S. N., Salzman, A. L., Boons, G. J., and Leibovich, S. J. (2003). An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. Am J Pathol 163, 711-721.
Radstake, T. R., Roelofs, M. F., Jenniskens, Y. M., Oppers-Walgreen, B., van Riel, P. L., Barrera, P., Joosten, L. A., and van den Berg, W. B. (2004). Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma. Arthritis Rheum 50, 3856-3865.
Ramanathan, M., Pinhal-Enfield, G., Hao, I., and Leibovich, S. J. (2007). Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter. Mol Biol Cell 18, 14-23.
Roelofs, M. F., Joosten, L. A., Abdollahi-Roodsaz, S., van Lieshout, A. W., Sprong, T., van den Hoogen, F. H., van den Berg, W. B., and Radstake, T. R. (2005). The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells. Arthritis Rheum 52, 2313-2322.
Schon, M. P., and Schon, M. (2008). TLR7 and TLR8 as targets in cancer therapy. Oncogene 27, 190-199.
Simmonds, R. E., and Foxwell, B. M. (2008). Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation. Rheumatology (Oxford) 47, 584-590.
Sumimoto, H., and Kawakami, Y. (2007). Lentiviral vector-mediated RNAi and its use for cancer research. Future Oncol 3, 655-664.
Suzuki, E., and Umezawa, K. (2006). Inhibition of macrophage activation and phagocytosis by a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin. Biomed Pharmacother 60, 578-586.
Traister, R. S., and Hirsch, R. (2008). Gene therapy for arthritis. Mod Rheumatol 18, 2-14.
Trinchieri, G., and Sher, A. (2007). Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 7, 179-190.
Tsai, C. Y., Shiau, A. L., Chen, S. Y., Chen, Y. H., Cheng, P. C., Chang, M. Y., Chen, D. H., Chou, C. H., Wang, C. R., and Wu, C. L. (2007). Amelioration of collagen-induced arthritis in rats by nanogold. Arthritis Rheum 56, 544-554.
Uematsu, S., and Akira, S. (2007). Toll-like receptors and Type I interferons. J Biol Chem 282, 15319-15323.
Uematsu, S., and Akira, S. (2008). Toll-Like receptors (TLRs) and their ligands. Handb Exp Pharmacol, 1-20.
Uprichard, S. L. (2005). The therapeutic potential of RNA interference. FEBS Lett 579, 5996-6007.
Wang, C. R., Chen, S. Y., Wu, C. L., Liu, M. F., Jin, Y. T., Chao, L., and Chao, J. (2005). Prophylactic adenovirus-mediated human kallistatin gene therapy suppresses rat arthritis by inhibiting angiogenesis and inflammation. Arthritis Rheum 52, 1319-1324.
Wang, C. R., Shiau, A. L., Chen, S. Y., Lin, L. L., Tai, M. H., Shieh, G. S., Lin, P. R., Yo, Y. T., Lee, C. H., Kuo, S. M., et al. (2008). Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer. Arthritis Rheum 58, 1650-1656.
Wiznerowicz, M., and Trono, D. (2003). Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 77, 8957-8961.
Zare, F., Bokarewa, M., Nenonen, N., Bergstrom, T., Alexopoulou, L., Flavell, R. A., and Tarkowski, A. (2004). Arthritogenic properties of double-stranded (viral) RNA. J Immunol 172, 5656-5663.
Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997). Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15, 871-875.