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系統識別號 U0026-2808201615412800
論文名稱(中文) 在前胸腺素轉殖基因小鼠中受損的傷口癒合
論文名稱(英文) Impaired Cutaneous Wound Healing in Prothymosin α Transgenic Mice
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 104
學期 2
出版年 105
研究生(中文) 蕭宜馨
研究生(英文) Yi-Hsin Hsiao
學號 S46034019
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-蕭璦莉
口試委員-吳昭良
口試委員-謝式洲
口試委員-歐弘毅
口試委員-李哲欣
中文關鍵字 傷口癒合  前胸腺素  乙型轉化生長因子 
英文關鍵字 wound healing  prothymosin α  TGF-β 
學科別分類
中文摘要 皮膚傷口癒合是一個複雜的過程,需要不同的介質,以及各種組織和細胞的協同作用,而正常的傷口癒合過程的失調可導致纖維化、傷痕形成,和組織功能的喪失,其中乙型轉化生長因子β(transforming growth factor-β, TGF-β)在傷口癒合和纖維化中扮演著重要的作用。前胸腺素( prothymosin α, ProT)是高度酸性蛋白,廣泛分佈於不同的細胞類型當中。根據先前實驗室的研究,我們發現ProT會抑制TGF-β的訊息傳遞,參與在肺氣腫疾病中的組織修復作用,以及參與在癌細胞當中之上皮細胞轉換成間質細胞,並且造成轉移的發生。因此,我們推測ProT在皮膚傷口癒合上,可能也會扮演著一定的角色。首先,我們收集了來自C57BL / 6小鼠在不同階段癒合中的傷口組織,ProT的mRNA會在癒合過程的早期階段(第3天)和重塑階段(第9天)高度表達。因此,我們接著比較ProT轉殖和野生型小鼠中之背部傷口手術之間的傷口組織,在ProT小鼠創傷修復後,其皮膚的膠原蛋白含量比較少,且其傷口組織中之磷酸化Smad2/3的蛋白也有較低的表現。因此我們推測在皮膚的傷口癒合當中,ProT轉殖小鼠可能因為其TGF-β訊息受到干擾,而引發了遲緩性的傷口癒合。免疫組織染色分析顯示,ProT過度表現的小鼠其生長後的傷口中,纖維母細胞特異蛋白S100A4以及其活化出的肌纖維母細胞特異性表現蛋白,α-平滑肌肌動蛋白(α-smooth muscle actin, α-SMA)的表達量皆比野生型的老鼠少。另外,和細胞外間質分解相關的抑制因子,金屬蛋白酶組織抑制劑TIMP-1,在ProT的小鼠傷口中的表現量也比較低。為了證實這些蛋白的表達與ProT之間的關係,我們利用染色質免疫沉澱的方式,發現ProT會藉由Smad7的乙烯化作用和α-SMA的啟動子結合,而Smad2與α-SMA啟動子的結合卻受到抑制,間接說明此蛋白的表達量因此受到阻礙。我們認為,其纖維母細胞相關的蛋白表達的減少,可能就是影響皮膚周圍細胞爬行到傷口進行修補能力降低的原因,而從細胞爬行的結果顯示,ProT的過度表達,確實會造成纖維母細胞以及角質細胞對於由TGF-β誘導的爬行能力受到抑制。綜合以上的結果,本篇研究說明ProT過度表達所造成的延緩性傷口癒合,是藉由增加Smad7對於和α-SMA的結合能力,抑制由TGF-β所誘導的爬行以及膠原蛋白的含量降低,最終導致傷口癒合的速度變慢。
英文摘要 Cutaneous wound healing is a complex process that requires collaborative efforts of different mediators as well as tissues and cell lineages. Deregulation of normal wound healing processes may result in fibrosis, scarring, and loss of tissue function. Transforming growth factor-β (TGF-β) plays an important role in orchestrating wound healing and fibrosis. Prothymosin α (ProT) is a highly acidic protein widely distributed in different cell types. Our previous studies have shown that ProT can antagonize TGF-β signaling, which is associated with empyema, resolution of liver fibrosis, and inhibition of metastasis of lung cancer. In this study, we hypothesized that ProT may be involved in cutaneous wound healing. First, we collected the wound tissue from C57BL/6 mice at different phases. ProT was highly expressed at the early stage (day 3) and remodeling stage (day 9) during the wound healing processes. Next, we compared the wound tissue between ProT transgenic (Tg) mice and wild-type (WT) mice undergoing dorsal wound surgery. We found that wound closure was delayed, and collagen deposition in the cutaneous wounds of both C57BL/6 and FVB ProT Tg mice was rescued. To understand the effect of regulation between ProT and TGF-β in the skin wound healing, immunohistochemical staining revealed that ProT was highly expressed in murine epidermis, and phosphorylated Smad2/3 was decreased in the wound tissue of ProT Tg mice. Furthermore, the fibroblast specific marker S100A4 and α-smooth muscle actin (α-SMA) were significantly decreased in ProT Tg mice. We also found that the inhibitor of matrix metalloproteinase-1 (TIMP-1) was decreased in the cutaneous wounds of ProT Tg mice. In addition, chromatin immunoprecipitation (ChIP) assay showed that the binding between Smad7 and the α-SMA promoter region was enhanced by ProT. Conversely, the binding of Smad2 to the α-SMA promoter was repressed by ProT. These results are in accordance with our previous findings that ProT can downregulate TGF-β-Smad signaling via Smad7 acetylation to affect gene transcription. We presumed that decreases in fibroblast-related markers may contribute to the reduction of recruitment of fibroblasts and keratinocytes and lead to the dysfunction of wound repair. Boyden chamber migration assay revealed impaired TGF-β-stimulated migratory activity in ProT-overexpressing cells. In conclusion, this study uncovers that excessive expression of ProT attenuates TGF-β signaling via Smad7 acetylation, thereby leading to impaired wound healing.
論文目次 Abstract......I
中文摘要......III
Acknowledgement......V
Content......VI
Figure Content......VIII
Introduction......1
1. Wound healing......1
1.1. Normal wound healing......1
1.2. Chronic wounds......1
2. Transforming growth factor-β (TGF-β) signaling......2
2.1. TGF-β......2
2.2. TGF-β signaling and Smads family......3
2.3. TGF-β signaling in cutaneous wound healing......3
3. Prothymosin α (ProT)......4
3.1. Biological roles of ProT......4
3.2 Role of ProT in regulation of acetylation......5
Specific Aim......6
Materials......7
I. Plasmids......7
II. Cell lines......7
III. Primers......8
IV. Reagents......8
V. Antibodies......9
VI. Bacteria......10
VII. Recombinant protein......10
VIII. Media and buffers......10
Methods......13
Cell cultures and treatments......13
Animal models......13
Isolation and treatment of mouse dermal fibroblasts....14
RNA extraction and reverse transcription......14
Real-time quantitative RT-PCR analysis (qRT-PCR)......14
Immunoblotting......15
Histological analysis......15
Lentivirus production......16
Chromatin immunoprecipitation (ChIP) assay......17
Boyden chamber migration assay......17
Results......19
Wound healing is impaired in the skin of ProT Tg mice..19
High expression of ProT contributes to down-regulation of TGF-β signaling......19
Accumulation of extracellular collagen is reduced in the wound tissue of ProT transgenic mice......19
Expression of fibroblasts and myofibroblast-related proteins is reduced in the wounds of ProT Tg mice......20
Expression of tissue inhibitors of metalloproteinases (TIMPs) is decreased in the wounds of ProT Tg mice.....20
ProT regulates Smad7-mediated transcriptional repression of the α-SMA gene by enhancing Smad7 acetylation......21
ProT interrupts TGF-β-mediated cells migration......22
Expression of ProT is higher in the genetically diabetic murine skin......23
STZ-treated mice exhibit lower body weight, higher fasting blood glucose levels, and larger wound size compared with control mice......23
Downregulation of dermal ProT improves STZ-induced delayed wound healing process......24
Discussion and Conclusions......25
1. Discussion......25
2. Conclusions......28
References......29
Figure legends......34
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