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系統識別號 U0026-2108201116224500
論文名稱(中文) 前胸腺素在轉型生長因子beta誘導之纖維母細胞活化中的影響
論文名稱(英文) Effects of prothymosin alpha on TGF-beta-induced fibroblast activation
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 99
學期 2
出版年 100
研究生(中文) 祁晨恩
研究生(英文) Bernard Kiro
學號 s46981012
學位類別 碩士
語文別 英文
論文頁數 41頁
口試委員 指導教授-蕭璦莉
口試委員-吳昭良
口試委員-李哲欣
口試委員-謝政蓉
口試委員-張孟雅
中文關鍵字 前胸腺素  纖維母細胞 
英文關鍵字 prothymosin  fibroblast 
學科別分類
中文摘要 前胸腺素 (Prothymosin alpha, ProT)最早是由大鼠的胸腺中分離出來的蛋白,而一開始是發現ProT是胸腺素alpha1 (Thymosin alpha1)的前驅物質,故稱之為前胸腺素。近幾年來陸陸續續有許多研究指出ProT會參與在許多細胞反應當中,其具有調節免疫反應、細胞增生、細胞凋亡、乙醯化作用、調節基因表現以及調控氧化壓力等功能。在腫瘤當中有百分之九十的體積是由細胞外基質和基質細胞所構成,而許多文獻也已經記載腫瘤細胞和細胞外基質之間的相互作用對於腫瘤的生長及疾病的進展是有高度相關性的。基質細胞包含了纖維母細胞、內皮細胞以及免疫細胞等等,其中以纖維母細胞對於整個腫瘤當中的細胞外基質的產生與分泌是有絕對關係的。過去的文獻已經證實將纖維母細胞給予TGF-beta的刺激會使纖維母細胞活化並且促使細胞外基質蛋白的合成與分泌。然而在我們的研究中發現ProT會經由穩定TGF-beta訊息傳遞的負調控因子Smad7去抑制TGF-的訊息傳遞。所以我們假設ProT會去抑制TGF-對纖維母細胞的活化。我們建立了ProT過度表現的纖維母細胞株NIH-3T3,並且去探討ProT對於纖維母細胞的TGF-訊息傳遞是否有抑制的現象。結果發現ProT會減少TGF-訊息下游的Smad2磷酸化,但同時增加Smad7的表現量,且纖維母細胞的活化指標SMA的表現也明顯受到抑制。我們也去探討了ProT對於細胞外基質蛋白的誘導作探討,結果發現在TGF-beta刺激下ProT會抑制纖維母細胞的fibronectin表現量,但卻可增強tenascin-C的表現,對於collagen type I、collagen type IV與laminin-gamma1的表現則沒有顯著影響。另外也偵測到ProT會增強TIMP-1表現,但同時抑制MMP-2基質蛋白脢表現。我們也利用wound healing assay觀察到了ProT會去抑制纖維母細胞的移動能力,也經由多光子共厄焦顯微鏡觀察到纖維母細胞與黑色素瘤細胞B16F10共同培養下,ProT會減少tenascin-C在兩種細胞接觸部位的沉積。最後,我們在ProT轉殖基因小鼠黑色素瘤模型中觀察到ProT轉殖基因小鼠的腫瘤是比野生型小鼠的腫瘤來的小的。綜合上述結果,體外實驗證明ProT會抑制纖維母細胞在TGF-刺激下的活化,如此可反應在ProT轉殖小鼠其纖維母細胞活性可能受到抑制而使腫瘤生長較緩慢,對於腫瘤治療上提供一種可能的方法。
英文摘要 Prothymosin  (ProT) was first isolated from rat thymus in 1984, and it was found to be the precursor of Thymosin 1, hence its name Prothymosin . Throughout the years, ProT has been reported to involve in cell proliferation, apoptosis, acetylation, gene regulation, reactive oxygen species regulation, and immunomodulation. Extracellular matrix (ECM) and embedded cells, such as fibroblasts, endothelial cells, and immune cells, constitutes up to 90 % of the tumor, and the interaction of tumor cells and ECM played an important role in the initiation, progression, and metastasis of tumor. Fibroblasts are the major source of extracellular matrix production and secretion, and it has been reported that treatment of TGF- can activate fibroblasts. In our research, we discovered that ProT can inhibit TGF- signaling through stabilizing smad7, an inhibitory smad of TGF- signaling. We hypothesized that ProT might inhibit fibroblast activation. ProT-overexpressing fibroblasts were established and the inhibitory effect of TGF- signaling was observed. ECM production and secretion were also determined. ProT did not greatly alter the protein expression pattern of most ECM. We also used confocal microscope to evaluate the secretion of ECM protein on the cell surface, and found that the secretion is decreased in ProT-overexpressing fibroblasts. ProT transgene mice were used to determine the effect of ProT on tumor progression. As we suspected, the tumor volume measured from ProT transgene mice was smaller compared to wild-type mice. Taken together, ProT might inhibit fibroblast activation and subsequently interfere with tumor growth.
論文目次 Chinese abstract............................ I
English abstract............................ III
Acknowledgement..........................IV
Contents.............................. V
Figure contents............................ VII
Abbreviation............................ VIII
Introduction
Prothymosin ........................... 1
Fibroblast............................. 2
Transforming growth factor ..................... 3
Extracellular matrix......................... 4
Tumor stroma........................... 5
Specific aim............................ 6
Materials and methods
Cell culture............................ 7
Lentivirus gene delivery system.................... 7
RNA isolation and RT-PCR...................... 8
ProT isolation........................... 9
Immunoblotting.......................... 10
Immunofluorescence........................ 10
Wound healing.......................... 11
Proliferation assay......................... 11
Statistical analysis......................... 12
Results
Establishment of prothymosin  overexpressing fibroblasts......... 13
Prothymosin  inhibited the TGF-/Smad signaling and differentiation in
fibroblasts............................ 13
Effect of ProT in fibroblast extracellular matrix production......... 14
The effect of ProT on fibroblast proliferation............... 15
The effect of ProT on fibroblast migration................ 15
The expression of ECM protein on cell surface after TGF- treatment..... 16
In vivo tumor model........................ 17
Discussion............................. 18
References............................. 22
Table............................... 30
Figures............................... 31
The author............................. 41
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