成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	陳芳旭 Chen, Fang-Hsu
論文名稱：	介白素二十九在成骨細胞與破骨細胞中的調控角色 The regulatory role of IL-29 on osteoblasts and osteoclasts
指導教授：	許育祥 Hsu, Yu-Hsiang
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	61
中文關鍵詞：	介白素二十九、骨免疫、成骨新生、破骨新生、骨平衡
外文關鍵詞：	IL-29, Osteoimmunology, Osteoblastogenesis, Osteoclastogenesis, Bone homeostasis
相關次數：	點閱：122 下載：2
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

骨免疫（osteoimmunology）是近年被提起的新學門，主要是探討免疫系統與骨平衡(bone homeostasis)間的關聯。骨平衡主要由成骨細胞新生（osteoblastogenesis）與破骨細胞新生(osteoclastogenesis)調控，一旦失衡會導致骨頭疾病包含骨質疏鬆或關節炎。介白素二十九（Interleukin-29 ; IL-29）屬於第三型干擾素群中一員，主要透過細胞膜上IL-28R1及IL-10R2這對接受器來傳遞訊號啟動生物功能。IL-29在過往被認為在宿主對抗微生物入侵中具有重要角色，此外IL-29在被病毒感染的細胞中表現量上升。過去文獻指出，IL-29的來源包含巨噬細胞、樹突細胞、周邊血液單核細胞。從病理學角度切入， IL-29參與類風溼性關節炎及退化性骨關節炎，但對於IL-29在生理上骨平衡中的角色仍是未知的。我們利用人類造骨細胞株(hFOB1.19)來釐清IL-29對於成骨細胞新生的影響，輔以細胞存活率分析方式探討IL-29在成骨細胞增殖中的角色，並利用即時核酸定量分析及鹼性磷酯酵素(ALP)染色來分析成骨細胞分化情形。我們研究發現IL-29可以促進造骨前驅細胞的增生；在成骨細胞分化的階段，IL-29於前期有促進相關轉錄因子的表現，然而鹼性磷酯酵素染色結果顯示IL-29可能抑制成骨細胞的分化與成熟。此外，在破骨細胞新生的部分，我們利用人類周邊血單核細胞及小鼠骨髓細胞以取得破骨細胞前驅細胞，並以抗酒石酸磷酸酶(TRAP)染色來標記破骨細胞，結果中發現IL-29會抑制破骨細胞的分化。而在成骨細胞與破骨細胞共培養的環境下，在環境中添加IL-29，其標定破骨細胞的TRAP顏色較淺，意味者破骨細胞數量較少。綜合上述結果，我們認為IL-29在骨平衡中，促進成骨細胞的增殖，而抑制破骨細胞形成。

Osteoimmunology is the study of the interaction between immune system and skeletal system through regulating inflammation and bone homeostasis. Bone homeostasis is maintained by the dynamic balance between osteoblastogenesis and osteoclastogenesis. Moreover, an imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Interleukin-29 (IL-29), a member of type 3 interferon family, signals through the IL-28R1 and IL-10R2 heterodimer receptor complex. IL-29 plays an important role in the host against microbes and expressed in those virus-infected cells. Immune cells, such as macrophages, dendritic cells and peripheral blood monocytes, are the cellular source of IL-29 Previous studies indicated that IL-29 modulates inflammatory response in rheumatoid arthritis and osteoarthritis. However, its biological role in bone homeostasis remains unclear. Our aims, therefore, were to explore whether IL-29 involved in the bone homeostasis through regulating osteoblast and osteoclasts. First, we found that osteoblast progenitor hFOB1.19 cell was the target for IL-29, and stimulation with IL-29 led to upregulation of proinflammatory cytokines. For the osteoblastogenesis, we analyzed both the process of proliferation and differentiation stage. IL-29 promoted osteoblast proliferation and early differentiation through upregulating osteoblastic gene Runx2 and Osterix. However, IL-29 inhibited ALP positive cells and RANKL gene expression on the late osteoblast differentiation. For osteoclastogenesis, we performed in vitro osteoclast differentiation system in both human and mouse, and found that IL-29 act as a negative regulator in osteoclasts differentiation. IL-29 decreased the numbers of osteoclasts, and reduced the TRAP and cathepsin K mRNA expression. To determine the role of IL-29 in the crosstalk between osteoblasts and osteoclasts, then we co-cultured with osteoblasts and osteoclasts. Our results showed that co-culture with IL-29 led to decreasing of TRAP purple color, which confirmed that IL-29 in the co-culture system ended up downregulated osteoclast differentiation. In this present study, we concluded that IL-29 expressed in human osteoblasts and promoted osteoblast proliferation. Also, IL-29 significantly inhibited osteoclast differentiation. With these findings and further elucidation of the pathways can provide more information to the development of novel agents for treating osteoporosis.

中文摘要 ................................I 
Abstract .............................. II 
Acknowledgement........................ IV 
Contents .............................. V 
Contents of Table...................... VIII 
Contents of Figure..................... IX 
Contents of Supplementary Appendix ..... X 
Abbreviation............................ XI 
Research motive ........................ XII 

I. Introduction ........................ 1 
1. Bone homeostasis .................... 1 
2. Cytokines ........................... 1 
3. Osteoimmunology...................... 2 
4. Interleukin-10 family................ 3 
5. Interleukin-29 ...................... 4 
6. Biological function of IL-29 ........ 5 
7. The role of IL-29 in osteoimmunology ... 6 

II. Materials and Methods ................. 7 
1. Human osteoblast differentiation........ 7 
2. Isolation of RNA ....................... 7 
3. Reverse-transcription-RCR (RT-PCR) ..... 7  
4. Quantification Real Time PCR (Real-time qPCR/RT-qPCR)..8 
5. Immunocytochemistry staining ........... 8 
6. Cell immunofluorescence ................ 9 
7. Cell proliferation assay ............... 9 
8. Alkaline phosphatase (ALP) staining .... 10 
9. Alizarin Red S (ARS) staining .......... 10 
10. Human Osteoclast differentiation ...... 11 
11. Mouse osteoclasts differentiation ..... 11 
12. TRAP staining ......................... 12 
13. Purification of recombinant human IL-29 (rhIL-29) from mammalian cells ....................... 12 
14. Purification of IL-29 monoclonal antibody.................................... 13 
15. Confirming protein and antibody expression using Coomassie blue and Western blotting .........13 
16. Functional assays for recombinant human IL-29 protein..................................... 14 
17. Statistical analysis ............................................ 14
 
III. Results ................................... 15 
1. Expression of IL-29 and its heterodimer receptors in hFOB1.19 cells ..................................... 15 
2. IL-29 modulated pro-inflammatory cytokine expression in hFOB1.19 cells .............................. 15 
3. IL-29 promoted hFOB1.19 cell proliferation ................................................ 15 
4. IL-29 reduced the ALP and RANKL mRNA level during the late differentiation ........................... 16 
5. IL-29 upregulated Runx2 expression during the differentiation at day 7 ....................... 16  
6. Stimulation with IL-29 increased pro-inflammatory cytokines gene expression in differentiated osteoblasts ................................................ 17 
7. Generation human and mouse osteoclasts ................................................ 17 
8. Expression of IL-29 receptors in human osteoclasts ................................................ 18 
9. IL-29 inhibited human osteoclast differentiation ................................................ 18 
10. IL-29 decreased human osteoclastic gene expression...................................... 19 
11. IL-29 monoclonal antibody promoted human osteoclast differentiation ................................ 19 
12. IL-29 monoclonal antibody neutralized IL-29-mediated the inhibition of osteoclast differentiation ................................................ 19 
13. IL-29 inhibited mouse osteoclast differentiation ................................................ 20 
14. IL-29 downregulated mouse osteoclastic gene expression ..................................... 20 
15. Effects of IL-29 on human osteoclast formation in co-culture model .................................. 20 

IV. Discussion ................... 22 
V.References...................... 25 
VI. Tables ....................... 31 
VII. Figures and figure legends .................................. 33 
VIII. Supplementary Appendix .................................. 55

                                    

1 Lee, S. & Margolin, K. Cytokines in Cancer Immunotherapy. Cancers (Basel) 3, 3856-3893, doi:10.3390/cancers3043856 (2011).
2 Meier, F. M., Frerix, M., Hermann, W. & Muller-Ladner, U. Current immunotherapy in rheumatoid arthritis. Immunotherapy 5, 955-974, doi:10.2217/imt.13.94 (2013).
3 Xu, L. et al. Interleukin-29 induces receptor activator of NF-kappaB ligand expression in fibroblast-like synoviocytes via MAPK signaling pathways. Int. J. Rheum. Dis. 18, 842-849, doi:10.1111/1756-185x.12747 (2015).
4 Xu, L. et al. Interleukin-29 Enhances Synovial Inflammation and Cartilage Degradation in Osteoarthritis. Mediators Inflamm. 2016, 9631510, doi:10.1155/2016/9631510 (2016).
5 Rosenberg, N., Rosenberg, O. & Soudry, M. Osteoblasts in Bone Physiology—Mini Review. Rambam Maimonides Med J 3, doi:10.5041/rmmj.10080 (2012).
6 Del Fattore, A., Teti, A. & Rucci, N. Bone cells and the mechanisms of bone remodelling. Front Biosci (Elite Ed) 4, 2302-2321 (2012).
7 Kong, Y. Y. et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397, 315-323, doi:10.1038/16852 (1999).
8 Hikita, A. et al. Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand. J. Biol. Chem. 281, 36846-36855, doi:10.1074/jbc.M606656200 (2006).
9 Takayanagi, H. et al. Induction and Activation of the Transcription Factor NFATc1 (NFAT2) Integrate RANKL Signaling in Terminal Differentiation of Osteoclasts. Dev. Cell 3, 889-901, doi:https://doi.org/10.1016/S1534-5807(02)00369-6(2002).
10 Yoshitake, F., Itoh, S., Narita, H., Ishihara, K. & Ebisu, S. Interleukin-6 directly inhibits osteoclast differentiation by suppressing receptor activator of NF-kappaB signaling pathways. J. Biol. Chem. 283, 11535-11540, doi:10.1074/jbc.M607999200 (2008).
11 Coondoo, A. CYTOKINES IN DERMATOLOGY — A BASIC OVERVIEW. Indian J. Dermatol. 56, 368-374, doi:10.4103/0019-5154.84717 (2011).
12 Turner, M. D., Nedjai, B., Hurst, T. & Pennington, D. J. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim. Biophys. Acta 1843, 2563-2582, doi:10.1016/j.bbamcr.2014.05.014 (2014).
13 Chou, C.-W., Lin, F.-C., Tsai, H.-C. & Chang, S.-C. The importance of pro-inflammatory and anti-inflammatory cytokines in Pneumocystis jirovecii pneumonia. Med. Mycol. 51, 704-712, doi:10.3109/13693786.2013.772689 (2013).
14 Schreiber, G. & Walter, M. R. Cytokine receptor interactions as drug targets. Curr. Opin. Chem. Biol. 14, 511-519, doi:10.1016/j.cbpa.2010.06.165 (2010).
15 Kopf, M., Bachmann, M. F. & Marsland, B. J. Averting inflammation by targeting the cytokine environment. Nature Reviews Drug Discovery 9, 703, doi:10.1038/nrd2805 (2010).
16 Lorenzo, J., Horowitz, M. & Choi, Y. Osteoimmunology: Interactions of the Bone and Immune System. Endocr. Rev. 29, 403-440, doi:10.1210/er.2007-0038 (2008).
17 Hodge, J. M., Kirkland, M. A. & Nicholson, G. C. Multiple roles of M-CSF in human osteoclastogenesis. J. Cell. Biochem. 102, 759-768, doi:10.1002/jcb.21331 (2007).
18 Suda, T. et al. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20, 345-357, doi:10.1210/edrv.20.3.0367 (1999).
19 Boyle, W. J., Simonet, W. S. & Lacey, D. L. Osteoclast differentiation and activation. Nature 423, 337-342, doi:10.1038/nature01658 (2003).
20 Takayanagi, H. et al. RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-beta. Nature 416, 744-749, doi:10.1038/416744a (2002).
21 Arron, J. R. & Choi, Y. Bone versus immune system. Nature 408, 535, doi:10.1038/35046196 (2000).
22 Ouyang, W., Rutz, S., Crellin, N. K., Valdez, P. A. & Hymowitz, S. G. Regulation and Functions of the IL-10 Family of Cytokines in Inflammation and Disease. Annu. Rev. Immunol. 29, 71-109, doi:10.1146/annurev-immunol-031210-101312 (2011).
23 Shouval, D. S. et al. Interleukin 10 Receptor Signaling: Master Regulator of Intestinal Mucosal Homeostasis in Mice and Humans. Adv. Immunol. 122, 177-210, doi:10.1016/B978-0-12-800267-4.00005-5 (2014).
24 Alexander, S. P. et al. THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G protein-coupled receptors. Br. J. Pharmacol. 174 Suppl 1, S17-s129, doi:10.1111/bph.13878 (2017).
25 Lauw, F. N. et al. Proinflammatory Effects of IL-10 During Human Endotoxemia. The Journal of Immunology 165, 2783-2789, doi:10.4049/jimmunol.165.5.2783 (2000).
26 Jain, S., Gabunia, K., Kelemen, S. E., Panetti, T. S. & Autieri, M. V. The Anti-Inflammatory Cytokine Interleukin-19 Is Expressed in and Angiogenic for Human Endothelial Cells. Arterioscler. Thromb. Vasc. Biol. 31, 167-175, doi:10.1161/atvbaha.110.214916 (2011).
27 Wei, C. C. et al. IL-20: biological functions and clinical implications. J. Biomed. Sci. 13, 601-612, doi:10.1007/s11373-006-9087-5 (2006).
28 Sheppard, P. et al. IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat. Immunol. 4, 63-68, doi:10.1038/ni873 (2003).
29 Kotenko, S. V. et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex. Nat. Immunol. 4, 69-77, doi:10.1038/ni875 (2003).
30 Hamming, O. J., Gad, H. H., Paludan, S. & Hartmann, R. Lambda Interferons: New Cytokines with Old Functions. Pharmaceuticals 3, 795-809, doi:10.3390/ph3040795 (2010).
31 Syedbasha, M. & Egli, A. Interferon Lambda: Modulating Immunity in Infectious Diseases. Front. Immunol. 8, doi:10.3389/fimmu.2017.00119 (2017).
32 Wolk, K. et al. Maturing dendritic cells are an important source of IL-29 and IL-20 that may cooperatively increase the innate immunity of keratinocytes. J. Leukoc. Biol. 83, 1181-1193, doi:10.1189/jlb.0807525 (2008).
33 Wolk, K. et al. IL-29 is produced by T(H)17 cells and mediates the cutaneous antiviral competence in psoriasis. Sci. Transl. Med. 5, 204ra129, doi:10.1126/scitranslmed.3006245 (2013).
34 Hung, C. H. et al. IL-28 and IL-29 as protective markers in subject with dengue fever. Med. Microbiol. Immunol., doi:10.1007/s00430-017-0498-x (2017)
35 Doyle, S. E. et al. Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes. Hepatology 44, 896-906, doi:10.1002/hep.21312 (2006).
36 Koch, S. & Finotto, S. Role of Interferon-lambda in Allergic Asthma. J. Innate Immun. 7, 224-230, doi:10.1159/000369459 (2015).
37 Witte, K. et al. Despite IFN-λ receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type III interferons: implications for therapeutic applications of these cytokines. Genes Immun. 10, 702, doi:10.1038/gene.2009.72 (2009).
38 Maher, S. G. et al. IFN-α and IFN-λ differ in their antiproliferative effects and duration of JAK/STAT signaling activity. Cancer Biol. Ther. 7, 1109-1115 (2008).
39 Wolk, K., Witte, K. & Sabat, R. Interleukin-28 and interleukin-29: novel regulators of skin biology. J. Interferon Cytokine Res. 30, 617-628, doi:10.1089/jir.2010.0064 (2010).
40 Kelm, N. E. et al. The role of IL-29 in immunity and cancer. Crit. Rev. Oncol. Hematol. 106, 91-98, doi:10.1016/j.critrevonc.2016.08.002 (2016).
41 Wang, F. et al. Interleukin-29 modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis. Arthritis Res. Ther. 14, R228, doi:10.1186/ar4067 (2012).
42 Xu, D. et al. IL-29 Enhances LPS/TLR4-Mediated Inflammation in Rheumatoid Arthritis. Cell. Physiol. Biochem. 37, 27-34, doi:10.1159/000430330 (2015).
43 McInnes, I. B. & Schett, G. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205-2219, doi:10.1056/NEJMra1004965 (2011).
44 Xu, L. et al. IL-29 enhances Toll-like receptor-mediated IL-6 and IL-8 production by the synovial fibroblasts from rheumatoid arthritis patients. Arthritis Res. Ther. 15, R170, doi:10.1186/ar4357 (2013).
45 Chen, D. et al. Osteoarthritis: toward a comprehensive understanding of pathological mechanism. Bone Research 5, 16044, doi:10.1038/boneres.2016.44 (2017).
46 Scanzello, C. R. & Goldring, S. R. The Role of Synovitis in Osteoarthritis pathogenesis. Bone 51, 249-257, doi:10.1016/j.bone.2012.02.012 (2012).
47 Liu, H., Luo, T., Tan, J., Li, M. & Guo, J. Osteoimmunology' Offers New Perspectives for the Treatment of Pathological Bone Loss. Curr. Pharm. Des., doi:10.2174/1381612823666170511124459 (2017).
48 Henriksen, K., Karsdal, M. A., Taylor, A., Tosh, D. & Coxon, F. P. Generation of human osteoclasts from peripheral blood. Methods Mol. Biol. 816, 159-175, doi:10.1007/978-1-61779-415-5_11 (2012).
49 Yen, M. L. et al. Multilineage differentiation and characterization of the human fetal osteoblastic 1.19 cell line: a possible in vitro model of human mesenchymal progenitors. Stem Cells 25, 125-131, doi:10.1634/stemcells.2006-0295 (2007).
50 Narisawa, S., Frohlander, N. & Millan, J. L. Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev. Dyn. 208, 432-446, doi:10.1002/(sici)1097-0177(199703)208:3<432::aid-aja13>3.0.co;2-1 (1997).
51 Paul, H., Reginato, A. J. & Schumacher, H. R. Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum. 26, 191-200 (1983).
52 Stashenko, P., Dewhirst, F. E., Rooney, M. L., Desjardins, L. A. & Heeley, J. D. Interleukin-1 beta is a potent inhibitor of bone formation in vitro. J. Bone Miner. Res. 2, 559-565, doi:10.1002/jbmr.5650020612 (1987).
53 Gilbert, L. et al. Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2alpha A) is inhibited by tumor necrosis factor-alpha. J. Biol. Chem. 277, 2695-2701, doi:10.1074/jbc.M106339200 (2002).
54 Liu, X. H., Kirschenbaum, A., Yao, S. & Levine, A. C. Cross-talk between the interleukin-6 and prostaglandin E(2) signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-{kappa}B (RANK) ligand/RANK system. Endocrinology 146, 1991-1998, doi:10.1210/en.2004-1167 (2005).
55 Vesprey, A. & Yang, W. Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts. Bio-protocol 6, e1836, doi:10.21769/BioProtoc.1836 (2016).
56 Park, K. Y., Li, W. A. & Platt, M. O. Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation. Integr. Biol. (Camb.) 4, 1459-1469, doi:10.1039/c2ib20197f (2012).
57 Ginaldi, L., Di Benedetto, M. C. & De Martinis, M. Osteoporosis, inflammation and ageing. Immunity & ageing : I & A 2, 14-14, doi:10.1186/1742-4933-2-14 (2005).
58 Mathavan, N., Turunen, M. J., Tagil, M. & Isaksson, H. Characterising bone material composition and structure in the ovariectomized (OVX) rat model of osteoporosis. Calcif. Tissue Int. 97, 134-144, doi:10.1007/s00223-015-9991-7 (2015).
59 Mühl, H. Pro-Inflammatory Signaling by IL-10 and IL-22: Bad Habit Stirred Up by Interferons? Front. Immunol. 4, 18, doi:10.3389/fimmu.2013.00018 (2013).
60 Iyer, S. S. & Cheng, G. Role of Interleukin 10 Transcriptional Regulation in Inflammation and Autoimmune Disease. Crit. Rev. Immunol. 32, 23-63 (2012).
61 P., D. R., Faruk, S., V., K. S. & Harold, D. The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain. J. Leukocyte Biol. 76, 314-321, doi:doi:10.1189/jlb.0204117 (2004).

校內：2023-08-29公開
校外：2023-08-29公開

簡易檢索 / 詳目顯示

相關論文