ROGDI是一個新發現的基因位於人類染色體16p13.3的位置，依照GOA資料庫，ROGDI和造血及正調節細胞增生有關。為了探討這個新基因在肺部纖維化的角色，我們建立了體內及體外纖維化模式。從老鼠氣管打入bleomycin，並且在不同天數犧牲老鼠以取得肺部組織。Rogdi及其他纖維化相關因子，如CCL2及TGF-β1在早期小於10天內就會增加表現；相對的，抗纖維化因子，如IL-10、IFN-γ及HO-1在後期大於10天後才會增加表現。而precursor miR-21會隨著肺部纖維化嚴重度增加而增表現。以bleomcyin處理人類胚胎纖維母細胞 (WI-38 cells) 會使細胞產生纖維化特性，並使ROGDI及precursor miR-21增表現。以ROGDI轉染人類胚胎纖維母細胞再處理bleomycin，會增加人類胚胎纖維母細胞α-SMA及precursor miR-21的產生。兩個和細胞增生相關的因子，Akt及Erk，在ROGDI轉染過後的人類胚胎纖維母細胞表現，均比轉染空載體的纖維母細胞表現來的強。由我們的實驗可以得知ROGDI在肺部纖維化有增表現，並使纖維母細胞產生纖維化表現型。此外ROGDI轉染人類胚胎纖維母細胞會使precursor miR-21表現增加，而primary miR-21表現卻不受影響。這樣的現象暗示ROGDI可能透過TGF-β-Smad路徑去影響miR-21的活化，調節點可能在轉錄後層次。Ω

ROGDI, a novel gene, locates on human’s chromosome 16p13.3. According to Gene Ontology Annotation database, ROGDI is related to hemopoiesis and positive regulation of cell proliferation. In order to investigate the function of this novel gene in pulmonary fibrosis, fibrotic models in vivo and in vitro were created. Mice which received single intra-tracheal bleomycin injection were sacrificed on various intervals. Rogdi and other pro-fibrotic mediators, including CCL2 and TGF-β1, were up-regulated in the early phase(< 10 days). On contrary, the anti-fibrotic mediators IL-10, IFN-γ and heme oxygenase(HO)-1 were up-regulated in the late phase(> 10 days). The precursor microRNA 21 (miR-21) was up-regulated as the fibrotic severity increased. The human embryonic fibroblasts(WI-38 cells) showed fibrogenic phenotype and up-regulation of precursor miR-21 and ROGDI after bleomycin treatment. Human embryonic fibroblasts transfected by coding sequence of ROGDI showed up-regulated precursor miR-21 and α-SMA compared to those transfected by empty vectors after bleomycin treatment. Two signaling molecules related to positive regulation of cell proliferation, Akt and Erk, showed over-expressed after ROGDI transfection and bleomycin treatment compared to those with empty vector transfection. Our results imply that ROGDI is up-regulated in pulmonary fibrosis and turns fibroblasts into fibrogenic phenotype through positive regulation of miR-21. The increase of precursor, but not primary miR-21, after ROGDI transfection and bleomycin treatment indicates that ROGDI may regulate the TGF-β signaling pathway in human embryonic fibroblasts. Our results support that ROGDI is a novel gene for pulmonary fibrosis and warrants for further investigation. Ω

中文摘要-----------------------------------------------------------i-ii
英文摘要---------------------------------------------------------iii-iv
簡介----------------------------------------------------------------1-4
材料和方法-----------------------------------------------------5-13
結果-------------------------------------------------------------14-16
討論-------------------------------------------------------------17-21
參考文獻-------------------------------------------------------22-28
圖表-------------------------------------------------------------29-40
附錄-------------------------------------------------------------41-51
Ω

Bitterman, P.B., Rennard, S.I., Keogh, B.A., Wewers, M.D., Adelberg, S., Crystal, R.G., 1986. Familial idiopathic pulmonary fibrosis. Evidence of lung inflammation in unaffected family members. N. Engl. J. Med. 314, 1343-1347
Borzone, G., Moreno, R., Urrea, R., Meneses, M., Oyarzun, M., Lisboa, C., 2001. Bleomycin-induced chronic lung damage does not resemble human idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med.163, 1648–1653
Chaudhary, N.I., Schnapp, A., Park, J.E., 2006. Pharmacologic differentiation of inflammation and fibrosis in the rat bleomycin model. Am. J. Respir. Crit. Care Med. 173, 769-776
Chua, F., Gauldie, J., Laurent, G.J., 2005. Pulmonary fibrosis. Searching for model answers. Am. J. Respir. Cell Mol. Biol. 33, 9-13
Croce, C.M., 2009. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 10, 704–714
Davis, B.N., Hilyard, A.C., Lagna, G., Hata1, A., 2008. SMAD proteins control DROSA-mediated microRNA maturation. Nature. 454, 56-61
Ding, Q., Gladson, C.L., Wu, H., Hayasaka, H., Olman, M.A., 2008. Focal adhesion kinase (FAK)-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAKdependent manner. J. Biol. Chem. 283, 26839–26849
Ellis, R.H., 1965. Familial incidence of diffuse interstitial fibrosis. Postgrad. Med. J. 41, 150-152
Grunert, S., Jechlinger, M., Beug, H., 2003. Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat. Rev. Mol. Cell Biol. 4, 657–665
Harrison, J.H.Jr., Lazo, J.S., 1988. Plasma and pulmonary pharmacokinetics of bleomycin in murine strains that are sensitive and resistant to bleomcyin-induced pulmonary fibrosis. J. Pharmacol. Exp. Ther. 247, 1052-1058
Horvath, I., Donnelly, L.E., Kiss, A., Paredi, P., Kharitonov, S.A., Barnes, P.J., 1998. Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase–1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax. 53, 668–672
Huges, E.W., 1965. Familial incidence of diffuse interstitial fibrosis. Postgrad. Med. J. 41, 150-152
Kattla, J.J., Carew, R.M., Heljic, M., Godson, C., Brazil, D.P., 2008. Protein kinase B/Akt activity is involved in renal TGFbeta1-driven epithelial-mesenchymal transition in vitro and in vivo. Am. J. Physiol. Renal Physiol. 295, 215-225
Kumarswamy, R., Volkmann, I., Thum, T., 2011. Regulation and function of miRNA-21 in health and disease. RNA Biology. 8, 1-8
Lama, V.N., Phan, S.H., 2006. The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond. Proc. Am. Thorac. Soc. 3, 373-376
Latronico, M.V., Condorelli, G., 2009. MicroRNAs and cardiac pathology. Nat Rev Cardiol. 6, 419–429
Lee, P.J., Alam, J., Sylvester, S.L., Inamdar, N., Otterbein, L., Choi, A.M., 1996. Regulation of heme oxygenase–1 expression in vivo and in vitro in hyperoxic lung injury. Am. J. Respir. Cell Mol. Biol. 14, 556–568
Liu, G., Friggeri, A., Yang, Y., Milosevic, J., Ding, Q., Thannickal, V.J., Kaminski, N., Abraham, E., 2010. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J. Exp. Med. 207, 1589-1597
Lu, F., Zander, D.S., Visner, G.A., 2002. Increased expression of heme oxygenase–1 in human lung transplantation. J. Heart Lung Transplant. 21, 1120–1126
Maestrelli, P., El Messlemani, A.H., De Fina, O., Nowicki, Y., Saetta, M., Mapp, C., Fabbri, L.M., 2001. Increased expression of heme oxygenase (HO)–1 in alveolar spaces and HO-2 in alveolar walls of smokers. Am. J. Respir. Crit. Care Med. 164, 1508–1513
Maestrelli, P., Paska, C., Saetta, M., Turato, G., Nowicki, Y., Monti, S., Formichi, B., Miniati, M., Fabbri, L.M., 2003. Decreased heme oxygenase–1 and increased inducible nitric oxide synthase in the lung of severe COPD patients. Eur. Respir. J. 21, 971–976
Moore, B.B., Kolodsick, J.E., Thannickal, V.J., Cooke, K., Moore, T.A., Hogaboam, C., Wilke, C.A., Toews, G.B., 2005. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am. J. Pathol. 166, 675-684
Moore, B.B., Paine III, R., Christensen, P.J., Moore, T.A., Sitterding, S., Ngan, R., Wilke, C.A., Kuziel, W.A., Toews, G.B., 2001. Protection of pulmonary fibrosis in the absence of CCR2 signaling. J. Immunol. 167, 4368-4377
Murphy, A., O’Sullivan, B. J., 1981. Familial fibrosing alveolitis. Isr. J. Med. Sci. 150, 204-209
Nakamura, T., Matsushima, M., Hayashi, Y., Shibasaki, M., Imaizumi, K., Hashimoto, N., Shimokata, K., Hasegawa, Y., Kawabe, T., 2011. Attenuation of transforming growth factor-β-stimulated collagen production in fibroblasts by quercetin-induced heme oxygenase-1. Am. J Respir. Cell Mol. Biol. 44, 614–620
No author listed, 2000. Idiopathic pulmonary fibrosis: diagnosis and treatment. Am. J. Respir. Crit. Care Med. 161, 646-664
Ota, T., Suzuki, Y., Nishikawa, T., et al., 2004. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36, 40-45
Pandit, K.V., Corcoran, D., Yousef, H., Yarlagadda, M., Tzouvelekis, A., Gibson, K.F., Konishi, K., Yousem, S.A., Singh, M., Handley, D., Richards, T., Selman, M., Watkins, S.C., Pardo, A., Ben-Yehudah, A., Bouros, D., Eickelberg, O., Ray, P., Benos, P.V., Kaminski, N., 2010. Inhibition and role of let-7d in idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 182, 220–229
Phan, S.H., 2002. The myofibroblast in pulmonary fibrosis. Chest. 122, 286S-289S
Raghu, G., Mageto, Y.N., 1996. Genetic predisposition of interstitial lung disease. In T. E. King, Jr,. and M. I. Schwarz editors. Interstitial lung disease, 3rd ed. B.C. Decker. Hamilton, ON, Canada. 119-134
Roy, S., Khanna, S., Hussain, S.R., Biswas, S., Azad, A., Rink, C., Gnyawali, S., Shilo, S., Nuovo, G.J., Sen. C.K., 2009. MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue. Cardiovasc. Res. 82, 21–29
Scotton, C.J., Chambers, R.C., 2007. Molecular targets in pulmonary fibrosis. The myofibroblast in focus. Chest. 132, 1311-1321
Sheppard, D., 2006. Transforming growth factor beta: a central modular of pulmonary and airway inflammation and fibrosis. Proc. Am. Thorac. Soc. 3, 413-417
Sime, P.J., Xing, Z., Graham, F.L., Csaky, K.G., Gauldie, J., 1997. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induced prolonged severe fibrosis in rat lung. J. Clin. Invest. 100, 768-776
Stefani, G., Slack, F.J., 2008. Small non-coding RNAs in animal development. Nat. Rev. Mol. Cell Biol. 9, 219–230
Thannickal, V.J., Toews, G.B., White, E.S., Lynch III, J.P., Martinez, F.J., 2004. Mechanisms of pulmonary fibrosis. Annu. Rev. Med. 55, 395–417
Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz, S., 2008. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature. 456, 980-984
Tomasek, J.J., Gabbiani, G., Hinz, B., Chaponnier, C., Brown, R.A., 2002. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, 349–363
Vallance, B.A., Gunawan, M.I., Hewlett, B., Bercik, P., Van Kampen, C., Galeazzi, F., 2005. TGFbeta1 gene transfer to the mouse colon leads to intestinal fibrosis. Am. J. physiol. Gastrointest. Liver Physiol. 289, 116-128
Watters, L.C., 1986. Genetic aspects of idiopathic pulmonary fibrosis and hypersensitivity pneumonitis. Semin. Respir. Med. 7, 317-325
Zhang, K., Rekhter, M.D., Gordon, D., 1994. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis: a combined immunohistochemical and in situ hybridization study. Am. J. Pathol. 145, 114–125
Zhou, H., Lu, F., Latham, C., Zander, D.S., Visner, G.A., 2004. Heme oxygenase–1 expression in human lungs with cystic fibrosis and cytoprotective effects against Pseudomonas aeruginosa in vitro. Am. J. Respir. Crit. Care Med. 170, 633–640 Ω