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：	陳世堯 Chen, Shih-Yao
論文名稱：	類風濕性關節炎的基因治療 gene therapy for rheumatoid arthritis
指導教授：	吳昭良 Wu, Chao-Liang
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學研究所 Department of Biochemistry
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	38
中文關鍵詞：	類風濕性關節炎、腺病毒、轉型生成因子貝塔、膠原蛋白刺激關節炎
外文關鍵詞：	adenovirus, rheumatoid arthritis, transforming growth factor-beta, collagen induced arthritis
相關次數：	點閱：63 下載：5
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

類風濕性關節炎是一種慢性的關節疾病。此種疾病的主要特徵是會產生具發炎侵略性的滑膜炎。在疾病的早期，滑液膜內會形成新血管，逐漸會有免疫細胞的浸潤現象，最後滑液膜細胞會增生，形成所謂的血管翳。血管翳一開始會覆蓋軟骨，而隨著疾病的越來越嚴重，血管翳會逐漸的侵蝕軟骨，最後導致關節的破壞。而在許多的實驗證據中也指出在類風濕性關節炎病人的關節內，血管新生的作用是導致血管翳形成的最主要原因。在本研究中，我們主要是利用腺病毒載體攜帶thrombospondin-1，(這個重組載體命名為rAd-TSP-1)來做類風濕性關節炎的治療。除了利用thrombospondin-1抑制血管新生的能力之外，它也是轉型生成因子貝塔(TGF-beta)的活化劑。在一些研究中也認為轉型生成因子貝塔在類風濕性關節炎中會扮演抑制發炎的角色。而在我們的結果中也發現到當在關節炎大鼠的踝關節中打入rAd-TSP-1後，類風濕性關節炎的症狀會趨緩下來，而相較於施打rAd-LacZ的控制組則沒有明顯的差異。而在我們的治療組中(施打rAd-TSP-1)相較於控制組(施打rAd-LacZ)的滑液膜組織中看到血管的數目會有減少的現象。我們甚至看到了在治療組中的血管翳內TGF-beta會表現在免疫細胞浸潤較多的區域中，相較於控制組的血管翳內TGF-beta會表現在免疫細胞浸潤較少的區域中。因此我們認為TSP-1的療效不僅在於它有抑制血管新生的能力之外，而且還可以活化TGF-beta，來抑制發炎反應以達到治療類風濕性關節炎的效果。

Rheumatoid arthritis (RA) is a chronic joint disease characterized by an inflammatory erosive synovitis. Early synovial changes include neovascularization, inflammatory cell infiltration, and synovial cell hyperplasia-- a formation of pannus. This pannus initially covers cartilage, which is gradually eroded as the disease progresses, and eventually leading to joint destruction. Some lines of evidence have shown that angiogenesis, the formation of new blood vessels, is necessary for pannus formation in the joints of severely affected RA patients. In this study, an adenoviral vector expressing thrombospondin-1 (TSP-1), designated “rAd-TSP-1”, was constructed for the RA therapy. TSP-1, apart from acting as an angiogenic inhibitor, is a strong activator of transforming growth factor-beta (TGF-beta), which plays an anti-inflammatory role in the progression of RA. Our results showed that intraarticular injection of rAd-TSP-1 into the ankle joints of arthritic rats reduced the severity of RA syndrome, compared with those treated with rAd-LacZ, the control virus. Down-regulation of vascular numbers within the synovial tissue was observed in the therapeutic groups of animals treated with rAd-TSP-1, but not in rAd-LacZ-treated group. Moreover, our results showed that TGF-beta was localized in the area of more lymphocyte infiltration within the pannus of the therapeutic group, in contrast to the control group in the area of less lymphocyte infiltration. Therefore, these data suggest that the therapeutic effects of TSP-1 in RA may be attributable to its direct anti-angiogenic effect but also to its indirect anti-inflammatory activity through the induction of TGF-beta.

Content

Chinese abstract……………………………I
Abstract………………………………………Ⅲ
Content……………………………………….Ⅳ
Figure content………………………………Ⅵ
Abbreviation…………………………………Ⅶ
Achknowledgement......................Ⅷ

Introduction
Rheumatoid arthritis………………………1
Gene therapy for RA……………………….2
Adenoviral vector………………………….3
Thrombospondin-1……………………......4
Transforming growth factor-beta……….5
Experimental strategies………………….6	

Materials and methods
Preparation of recombinant adenovirus.7
Animal studies………………………………7
Histological processing of the ankle joints…..............................8
Immunohistochemistry analysis………….9
Statistics analysis……………………….10	

Results
Expression of TSP-1 in A549 cells infected with rAd-TSP-1…...........................11
Arthritis evaluation by collagen immunization……......................11
Marked vascularity in rats with CIA………………………………...........12
Expression of the transferred gene and macroscopic effects on 
CIA...................................12
Suppression of CIA by TSP-1 gene transfer………………………............13
Reduction of angiogenesis after TSP-1 gene transfer…………......................14
Different localization of TGF-beta after TSP-1 gene transfer.........................14	


Discussion……………………………………16
References……………………………………19
Appendix.1……………………………………35
Appendix.2……………………………………36
Appendix.3……………………………………37

Figure content

Fig.1.	Expression of TSP-1 in A549 cells infected with rAd-TSP-1….............25

Fig.2.	Arthritis induction by intradermal injection with bovine type II collagen in SD rats……………………………………………26

Fig.3.	Radiography for the normal and arthritic joints………………..........27

Fig.4.	Marked vascularity in ankle joints of rats with collagen-induced arthritis (CIA)…...………………………………..............28

Fig.5.	Expression of TSP-1 in the synovial tissue infected by rAd-TSP-1…........29

Fig.6.	TSP-1 gene transfer prevented swelling of the ankle in the animal model of RA……………………………………………..30

Fig.7.	Recovery of gait pattern followed by TSP-1 gene therapy……................31

Fig.8.	Effects of TSP-1 gene therapy on pannus formation and mononuclear cell infiltration in collagen-induced arthritis………......32

Fig.9.	Effects of TSP-1 gene therapy on arthritis-associated angiogenesis………………………………….33

Fig.10.	Different localization of TGF- in the RA synovium…………....................34

Appendix1.Comparison of normal and rheumatoid joints………….........................35

Appendix2.Structure and functional domain of thrombospondin-1 (TSP-1)...............36

Appendix3.The strategy for producing replication-defective adenovirus (AdEasy-1 system)………………………………………………...37

                                    

(1) Zvaifler NJ, Firestein GS. Pannus and pannocytes. Alternative models of joint destruction in rheumatoid arthritis. Arthritis Rheum 1994; 37:783-789.
(2) Apparailly F, Verwaerde C, Jacquet C, Auriault C, Sany J, Jorgensen C. Adenovirus-mediated transfer of viral IL-10 gene inhibits murine collagen-induced arthritis. J Immunol 1998; 160:5213-5220.
(3) Lubberts E, Joosten LA, van Den BL, Helsen MM, Bakker AC, van Meurs JB et al. Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction. J Immunol 1999; 163:4546-4556.
(4) Kimball ES, Gross JL. Angiogenesis in pannus formation. Agents Actions 1991; 34:329-331.
(5) Paleolog EM. Angiogenesis in rheumatoid arthritis. Arthritis Res 2002; 4:S81-S90.
(6) Colville-Nash PR, Scott DL. Angiogenesis and rheumatoid arthritis: pathogenic and therapeutic implications. Ann Rheum Dis 1992; 51:919-925.
(7) Koch AE. Review: angiogenesis: implications for rheumatoid arthritis. Arthritis Rheum 1998; 41:951-962.
(8) Fava RA, Olsen NJ, Spencer-Green G, Yeo KT, Yeo TK, Berse B et al. Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med 1994; 180:341-346.
(9) Kim JM, Ho SH, Park EJ, Hahn W, Cho H, Jeong JG et al. Angiostatin gene transfer as an effective treatment strategy in murine collagen-induced arthritis. Arthritis Rheum 2002; 46:793-801.
(10) Yin G, Liu W, An P, Li P, Ding I, Planelles V et al. Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis. Mol Ther 2002; 5:547-554.
(11) Hollander AP, Corke KP, Freemont AJ, Lewis CE. Expression of hypoxia-inducible factor 1alpha by macrophages in the rheumatoid synovium: implications for targeting of therapeutic genes to the inflamed joint. Arthritis Rheum 2001; 44:1540-1544.
(12) Heise C, Kirn DH. Replication-selective adenoviruses as oncolytic agents. J Clin Invest 2000; 105:847-851.
(13) Yu, H., Giai, M., Diamandis, E. P., Katsaros, D., Sutherland, D. J., Levesque, M. A., Roagna, R., Ponzone, R. Sismondi, P. Prostate-specific antigen is a new favorable prognostic indicator for woman with breast cancer. Cancer Research 1995; 55:2104-2110.
(14) Zarghami, N., Grass, L., Diamandis, E. P. Steroid hormone regulation of prostate-specific antigen gene expression in breast cancer. British Journal of Cancer 1997; 75:579-588.
(15) Crystal, R. G. Transfer of genes to humans: early lessons and obstacles to success. Science 1995; 270:404-410.
(16) Lawler JW, Slayter HS, Coligan JE. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J Biol Chem 1978; 253:8609-8616.
(17) Taraboletti G, Roberts D, Liotta LA, Giavazzi R. Platelet thrombo- spondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J Cell Biol 1990; 111:765-772.
(18) Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci U S A 1990; 87:6624-6628.
(19) Dameron KM, Volpert OV, Tainsky MA, Bouck N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 1994; 265:1582-1584.
(20) Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, Bouck N. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol 1993; 122:497-511.
(21) Schultz-Cherry S, Chen H, Mosher DF, Misenheimer TM, Krutzsch HC, Roberts DD et al. Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1. J Biol Chem 1995; 270:7304-7310.
(22) Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP. CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells. J Cell Biol 1997; 138:707-717.
(23) Jimenez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 2000; 6:41-48.
(24) Chung J, Gao AG, Frazier WA. Thrombspondin acts via integrin–associated protein to activate the platelet integrin alphaIIbbeta3. J Biol Chem 1997; 272:14740-14746.
(25) Schultz-Cherry S, Lawler J, Murphy-Ullrich JE. The type 1 repeats of thrombospondin 1 activate latent transforming growth factor-beta. J Biol Chem 1994; 269:26783-26788.
(26) Schultz-Cherry S, Murphy-Ullrich JE. Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J Cell Biol 1993; 122:923-932.
(27) Song XY, Gu M, Jin WW, Klinman DM, Wahl SM. Plasmid DNA encoding transforming growth factor-beta1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model. J Clin Invest 1998; 101(12):2615-2621.
(28) Wahl SM. Transforming growth factor beta (TGF-beta) in inflammation: a cause and a cure. J Clin Immunol 1992; 12:1-14.
(29) Palmblad K, Erlandsson-Harris H, Tracey KJ, Andersson U. Dynamics of early synovial cytokine expression in rodent collagen-induced arthritis: a therapeutic study using a macrophage-deactivating compound. Am J Pathol 2001; 158:491-500
(30) He, T. C., Zhou, S., Yu, J., Kinzler, K. W., Vogelstein, B. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 1998; 95:2509-2514.
(31) Woods JM, Katschke KJ, Volin MV, Ruth JH, Woodruff DC, Amin MA et al. IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis. J Immunol 2001; 166:1214-1222.
(32) Nicosia RF, Tuszynski GP. Matrix-bound thrombospondin promotes angiogenesis in vitro. J Cell Biol 1994; 124:183-193.
(33) Weinstat-Saslow DL, Zabrenetzky VS, VanHoutte K, Frazier WA, Roberts DD, Steeg PS. Transfection of thrombospondin-1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 1994; 54:6504-6511.
(34) Koch AE, Szekanecz Z, Friedman J, Haines GK, Langman CB, Bouck NP. Effects of thrombospondin-1 on disease course and angiogenesis in rat adjuvant-induced arthritis. Clin Immunol Immunopathol 1998; 86:199-208.

校內：2005-09-04公開
校外：2005-09-04公開

簡易檢索 / 詳目顯示

相關論文