臺灣博碩士論文加值系統

中文摘要
蟹足腫是人類常見的疾病之一，是一種過度增生的疤痕組織，患者於創傷後，癒合的傷口超過原本皮膚的高度及傷口範圍，侵犯到周圍的正常組織。治療上相當困難，以傳統治療（手術及病灶內類固醇注射），復發率高達百分之五十。本病的制病機轉仍未明，細胞外成纖維細胞對細胞激素的反應及其下游的訊息傳遞基質膠原蛋白聚積可能為重要原因。
乙型轉型生長因子（TGF-β）可促進成纖維細胞的移行、增殖和膠原蛋白的製造，在許多慢性纖維化疾病當中，佔有相當重要的角色。
TGF-β主要相關的細胞內訊息傳導物質為Smads蛋白質，分為活化型轉錄因子Smad2、Smad3 和一般型轉錄因子Smad4及抑制型轉錄因子Smad6、Smad7。本實驗利用正常與蟹足腫成纖維細胞株施以各種濃度組合之TGF-β1投與方式，期望能找出其細胞內訊息傳遞及蛋白質的釋出之差異。
研究結果顯示正常成纖維細胞株之Smad2、Smad3、Smad4的表現沒有明顯的增加，而Smad6、Smad7有明顯增加。而蟹足腫成纖維細胞株的Smad2的表現有明顯的增加，Smad3、Smad4的表現沒有明顯的增加，Smad6、Smad7則沒有表現。綜合以上結果顯示乙型轉型生長因子對正常成纖維細胞株的作用是經由增加抑制型轉錄因子Smad6、Smad7來抑制活化型轉錄因子Smad2、Smad3，使之表現正常；而乙型轉型生長因子對蟹足腫成纖維細胞株而言，則是不經抑制型轉錄因子Smad6、Smad7的抑制調控，直接刺激活化型轉錄因子Smad2表現增加，故推測蟹足腫病變可能透過此一訊息傳遞之表現而增加細胞外基質蛋白的合成與堆積。

Abstract:Keloid is one of the most common skin disease in human beings.Which defined as scars growing beyond the confines of original woundsafter healing of a skin injury. There is no universally accepted effective treatment, with the conventional therapy, i.e. surgery and/or intralesional steroid injection，the recurrence rate is 50﹪.The pathogenesis is still unknow. The abnormalities in extracellular matrix collagen production, abnormal response of the fibroblasts to various cytokines and the signal transduction might be the important factors.
TGF-β can induce cell migration、proliferation and production of collagen in fibroblasts, and play a key role in many chronic fibrotic disorders. Previous investigations revealed that Smads were involved in the TGF-β signaling in human fibroblasts. There are several homologs of Smad protein in the cell. Smad2 and Smad3 are receptor-regulated Smads .and Smad4 as the common mediator Smad and Smad6、7 as inhibitory Smads. The purpose of this study was designed by using the CCD966SK and KEL FIB cultures treated with different concentration of TGF-β1 to discover the regulatory mechanisms and the difference amount of releasing protein and gene expression.
As the result, we discovered that in CCD966SK there was no apparent increased amount in Smad2, Smad3 and Smad4 expressed, but Smad6
、7 showed a significant increasing amount. For KEL FIB, the expression of Smad2 could be detected with increasing amount, but Smad3 and Smad4 had shown in small increasing amount. In contrast, Smad6 and Smad7 had no expression showed in this experiment. In conclusion, the effects of TGF-β on CCD966SK are in turn mediated by inhibition of Smad2 and Smad3 due to increase in Smad6 and Smad7. The effects of TGF-β on KEL FIB are in turn mediated activation of Smad2 without Smad6 and Smad7 regulation control. Therefore, the formation of Keloid
might be caused by the regulatory mechanisms of TGF-β which stimulated extracellular matrix synthesis and deposition.

目 錄
一、 中文摘要………………………………………. 1
二、 英文摘要………………………………………. 3
三、 前言……………………………………………. 5
四、 材料與實驗方法………………………………. 20
五、 研究結果………………………………………. 27
六、 討論……………………………………………. 33
七、 未來展望………………………………………. 38
八、 圖表……………………………………………. 39
九、 參考文獻………………………………………. 51

Border WA,Ruoslahti E:Transforming growth factor-β in disease:the dark side of tissue repair. J Clin Invest90:1-7,1992.
Boyadjiev C, Pppchristora E, Mazgalova J: Histomorphologic changes in keloids treated with kenacort. J Traum 38:299-302,1994.
Brodin G, Dijke PT, Funa K, Heldin CH and Landstrom M: Increased Smad expression and activation are associated with apoptosis in normal and malignant prostate after castration. Cancer Res 59: 2731-2738,1999.
Bruno E, Horrigan SK, Berg D, Rozler E, Fitting PR, Moss St, Westbrook C and Hoffman R: The Smads gene is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-β on human hematopoiesis. Blood 91: 1917-1923,1998.
Caestecker MP, hemmati P, Larisch-Bloch S, Ajmera R, Roberts AB and Lechleider RJ: Characterization of functional domains within Smad4/DPC4. J Biol Chem 272: 13690-13696,1997.
Calonge MJ and Massague J: Smad4/DPC4 silencing and hyperactive Ras jointly disrupt transforming growth factor-β antiproliferative responses in colom cancer cells. J Biol Chem 274: 33637-33643,1999.
Candia AF, Watabe T, Hawley SHB, Onichtchouk D, Zhang Y, Derynck R, Niehrs C and Cho KWY: Celluar interpretation of multiple TGF-β signals: intracellular antagonism between activin/BVgl and BMP-2/4 signalling mediated by Smads. Development 124: 4467-4480,1997.
Carcamo J, Weis FM, Ventura F, Wieser R, Wrana JL, Attisano L, Massague J: Type I receptors specify growth-inhibitory and transcription response to transforming growth factor beta and activin. Mol Cell Biol 14: 3810-3421,1994.
Chen RH, Ebner R, Derynck R: Inactivation of type II receptor reveals two receptor pathways for the diverse TGF-beta activities. Science 260: 1335-1338,1993.
Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M and Varga J: Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad3. J Invest Dermatol 112: 49-57,1999.
Chen wei, Fu Xiaobing, Sun Tongzhu, Sun Xiaoqing, Zhao Zhili, Sheng Zhiyong:Change of gene expression of transforming growth factor-β1,
Smad2 and Smad3 in hypertrophic scars skins. Chin J Surg 40:17-19,2002.
Chen Y, Bhushan A and Vale W: Smad8 mediates the signalling of the receptor serine kinase. Proc Natl Acad Sci 94: 12938-12943,1997.
Chin GS, Liu W, Peled Z, Lee TY, Steinbrech DS, Hsu M, Longaker MT: Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts. Plast Reconstr Surg.108:423-9, 2001.
Das P, Maduzia LL, Wang H, Finelli AL, Cho SH, Smith MM and Padgett RW: The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling. Development 125: 1519-1528,1998.
Datubo-Brown DD.Keloids: a review of the literature. Br J Plast Surg 43: 70-77,1990.
Dennler S, Itoh S, Vivien D, Dijke PT, Huet S and Gauthier JM: Direct binding of Smad3 and Smad4 to critical TGFβ-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. The EMBO J 17: 3091-3100,1998.
Dennler S, Huet S and Gauthier JM: Aa short amion-acid sequence in MH1 domain is responsible for functional differences between Smad2 and Smad3. Oncogene 18: 1643-1648,1999.
Derynck R: TGF-beta-receptor-mediated signalling. Trend Biochem Sci 19: 548-553,1994.
Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC and Lan HY: Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 56: 1455-1467,1999.
Feng XH, ZhangY, Wu RY and Derynck R: The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for Smads in TGF-β-induced transcriptional activation: Genes 12:2153-63,1998.
Gesualdo L, Ranieri E, Monno R, Rossiello MR, Colucci M, Semeraro N, Grandaliano G and Schena FP: Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells. Kidney int 56: 461-470,1999.
Goto D, Yagi K, Inoue H, Iwamoto I, Kawabata M, Miyazono K and Kato M: A single missense mutant of Smad3 inhibits activation of both Smad2 and Smad3, and has a dominant negative effect on TGF-β signals. FEBS 430: 201-204,1998.
Hanasono MM, Kita M, Mikulec AA, Lonergan D, Koch RJ: Autocrine growth factor production by fetal, keloid, and normal dermal fibroblasts. Arch Facial Plast Surg..5:26-30, 2003.
Hayadhi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA, Wrana JL, and Falb D: The MAD-related protein Smad7 associated with the TGFβ receptor and Functions as an antagonist of TGFβ signalling. Cell 89: 1165-1173,1997.
Heldin CH, Miyazono K and Dijke PT: TGF-β signalling from cell membrane to nucleus through Smad proteins. Nature 390: 465-471,1997.
Heldin NE, Bergstrom D, Hermansson A, Bergenstrahle A, Nakao A, Westermark B and Dijke PT: Lack of responsiveness to TGF-β1 in a thyroid carcinoma cell line with functional type I and type II TGF-β receptors and Smad proteins, suggests a novel mechanism for TGF-β insensitivity in carcinoma cells. Mol Cell Endocrin 153: 79-90,1999.
Hocevar BA, Howe PH: Mechanisms of TGF-β-induced cell cycle arrest. Miner Electrolyte Metab 24: 131-135,1998.
Horie K, Yamashita H, Mogi A, Takenoshita S and Miyazono K: Lack of transforming growth factor-β type II receptor expression in human eetinoblastoma cells. J Cell Physio 175: 305-313,1998.
Hua X, Liu X, Ansari DO, and Lodish HF: Synergistic cooperation of TFE3 and Smad proteins in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene. Genes and Deve 12: 3084-3095,1998.
Imamura T, Takase M, Nishihara A, Oeda E, Hanai JI, Kawabata M and Miyazono K: Smad6 inhibits signalling by the TGF-β superfamily. Nature 389: 622-626,1997.
Jahnson K, Kirkpatrick H, Comer A, Hoffmann FM and Laughon A: Interaction of Smad complexes with tripartite DNA-binding Site. J Biol chem 274: 20709-20716,1999.
Janknecht R, Wells NJ and Hunter T: TGF-β-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes and Development 12: 2114-2119,1998.
Jonk LJC, Itoh S, Heldin CH, Dijke PT and Kruijer W: Identification and functional characterization of a Smad binding element(SBE)in the Junb promoter that acts as a transforming growth factor-β, activin, and bone morphogenetic protein-inducible enhancer. J Biol Chem 273: 21145-21152,1998.
Kawabata M, Inoue H, Hanyu A, Imamura T and Miyazono K: Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/ threonine kinase receptors. EMBO J 17: 4056-65,1998.
Kischer CW, Hendrix MJ: Fibronectin (FN) in hypertrophic scars and keloids. Cell Tissue Res;231:29-37,1983.
Kleeff J, Ishiwata T, Maruyama H, Friess H, Truong P, Buchler MW, Falb D, and Korc M: The TGF-β signalling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer. Oncogene 18: 5363-5372,1999.
Korchynskyi O, Landstrom M, Stoika R, Funa K, Heldin CH, Duke PT and Soucheinytskyi S: Expression of Smad proteins in human colorectal cancer. Int. Journal of Cancer 82: 197-202,1999.
Kretzschmar M, Doody J and Massague J: Oppoaing BMP and EGF signalling pathways converge on the TGF-β family mediator Smad1. Nature 389-622 , 1997.
Lee KS.Song JY. Suh MH:Collagen mRNA expression detected by in situ beridisation in keloid tissue.J Dermatol Sci 316-323,1991.
Liberati NT, Datto MB, Frederick JP, Shen X, Wong C, Rougier-Chapman EM and Wang XF: Smads bind directly to the Jun family of AP-1 transcription factors. Proc. Natl. Acad. Sci.: 4819-4844,1999.
Luo K, Stroschein SL, Wang W, Chen D, Martens E, Zhou S and Zhou Q: The Ski oncoprotein interacts with the proteins to repress TGFβ signalling. Genes and Development 13: 2196-2206,1999.
Massague J: TGF-β signal transduction. Annu. Rev Biochem: 67: 753-791,1998.
Moustakas A and Kardassis D: Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members. Proc. Natl. Acad. Sci. 95: 6733-6738,1998.
Mucsi I and Goldberg HJ: Dominant-negative Smad3 interferes with transcriptional activation by multiple agonists. Biochem Biophys Res Commun 232: 517-521,1997.
Nagarajan RP, Zhang J, Li W and Chen Y : Regulation of Smad7 promoter by direct association with Smad3 and Smad4. J Biol Chem 274: 33412-33418, 1999.
Nakao A, Imamura T, Soucheinytskyi S, Hawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai JI, Heldin CH, Miyazono K and Dijke PT: TGF-β receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16: 5353-5362,1997.
Nakao A, Afrakhte M, Moren Aa, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Henrik CH and Dijke PT: Indentification of Smad7, a TGF-β-inducible antagonist of TGF-β signalling. Nature 389: 631-635,1997.
Nakayama T, Snyder MA, Grewal SS, Tsuneizumi K, Tabata T and Christian JL: Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning. Development 125: 857-867,1998.
Nowak KC, McCormack M, Koch RJ: The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors: a serum-free study. Plast Reconstr Surg 105:2039-48, 2000
Russell SB, Trupin JS, Kennedy RZ, Russell JD, Davidson JM: Glucocorticoid regulation of elastin synthesis in human fibroblasts: down-regulation in fibroblasts from normal dermis but not from keloids.
J Invest Dermatol 104:241-5,1995.
Sekelsky JJ, Newfeld SJ, Raftery LA, Chartoff EH, Gelbart WM: Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 139:
1347-58,1995.
Shi Y, Wang YF, Jayaraman L, Yang H, Massague J and Pavletich NP: Crystal structure of a Smad MH1 domain bound to DNA: Insights on DNA binding in TGF-β signalling. Cell 94: 585-594,1998.
Tabibzadeh S: Homeostasis of extracellular matrix by TGF-beta and lefty.Front Biosci 1:1231-46, 2002
Topper JN, Dichiara MR, Brown JD, Williams AJ, Falb D, Collin T and Gimbrone MA: CERB binding protein is a required coactivator for Smad-dependent, transforming growth factor β transcriptional responses in endothelial cells. Proc. Natl. Acad. Sci. 95: 9506-9511,1998.
Tsukazaki T, Chiang TA, Davison AF, Attisano L and Wrana JL: SARA, a FYVE domain protein that recruits Smad2 to the TGFβ receptor. Cell 95: 779-791,1998.
Usui T, Takase M, Kaji Y, Suzuki K, Ishida K, Tsuru T, Miyata K, Kawabata M and Yamashita H: Extracellular matrix production regulation by TGF-β in corneal endothelial cells. Invest Ophthalmol Vis Sci 39: 1981-1989,1998.
Uitto J, Perejda AJ, Abergel RP, Chu ML, Ramirez F: Altered steady-state ratio of type I/III procollagen mRNAs correlates with selectively increased type I procollagen biosynthesis in cultured keloid fibroblasts. Proc Natl Acad Sci 82:5935-5939,1985.
Verschueren K, Huylebroeck D: Remarkable versatility of Smad proteins in the nucleus of transforming growth factor-beta activated cells. Cytokine Growth Factor Rev10:187-99,1999.
Weinstein M, Yang X, Li C, Xu X, Gotay J, Deng CX: Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2. Proc Natl Acad Sci 95:9378-83,1998.
Weitkamp B, Gullen P, Plena G, Robenek H and Rauterberg J: Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque. FASEB J 13: 1445-1457,1999.
Whitman M: Feedback from inhibitory Smads. Nature 389: 549-551,1997.
Wong C, Rougier-Chapman EM, Frederick JP, Datto MB, Liberati NT, Li JM, Wang XF: Smad3-Smad4 and AP-1 complexes synergize in transcriptional activation of the c-Jun promoter by transforming growth factor beta. Mol Cell Biol 19: 1821-1830,1999.
Wotton D, Lo RS, Lee Sand Massague J: A Smad transcriptional corepressor. Cell 97: 29-39,1999.
Wrana J and Pawson T: Mad about Smads. Nature 388: 28-29,1997.
Wrana JL: TGF-β Receptors and Signalling mechanisms. Miner Electrolyte Metab 24: 120-130,1998.
Yeo CY, Chen X and Whitman M: The role of FAST-1 and Smads in transcriptional regulation by activin during early Xenopus embryogenesis. J Biol Chem 274: 26584-90,1999.
Zimmerman CM, Kariapper MST and Mathews LS: Smad proteins physically interact with calmodulin. J Biol Chem 273: 677-680 ,1998.
Zhang Y, Feng XH and Derynck R: Smad3 and Smad4 cooperate with c-Jun/c-Fos mediate TGF-β-induced transcription. Nature 394: 909-913,1998.
Zhou LJ, Ono I, Kaneko F: Role of transforming growth factor-beta 1 in fibroblasts derived from normal and hypertrophic scarred skin. Arch Dermatol Res 289:646-52, 1997.
Zhu H, Havsak P, Abdollah S, Wrana JL and Thomson GH: A Smad ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 400 : 687-693,1999.