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系統識別號 U0026-0808201211204700
論文名稱(中文) 探討凝血酶調節素在糖尿病傷口癒合中扮演的角色
論文名稱(英文) The role of thrombomodulin in diabetic wound healing
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 黃韋愷
研究生(英文) Wei-Kai Huang
電子信箱 k207791@hotmail.com
學號 s16994132
學位類別 碩士
語文別 英文
論文頁數 60頁
口試委員 指導教授-施桂月
口試委員-吳華林
口試委員-林淑華
口試委員-蔡曜聲
中文關鍵字 糖尿病  傷口癒合  凝血酶調節素 
英文關鍵字 diabetes  wound healing  Thrombomodulin 
學科別分類
中文摘要 糖尿病是由於血糖調控失衡造成的疾病,而長期處在高血糖下的糖尿病病人會引起許多併發症,包括發炎、 內皮細胞失去功能、以及慢性傷口等。其中傷口難以癒合的問題是普遍存在的。傷口造成後,角化細胞的爬行及生長促使表皮細胞癒合,形成一道抵禦外在環境刺激的屏障,此過程是傷口癒合中重要的一個環節。凝血酶調節素是一個表現在內皮及表皮細胞上的抗凝血醣蛋白,然而它在表皮細胞上的功能仍尚未清楚。在本篇研究中發現,有較多的凝血酶調節素會表現在傷口形成後第三天及第五天高度增生的上皮層當中,然而這個現象在誘發糖尿病的老鼠中會有表現延遲的情形。此外,在高度增生上皮層中,正在生長的角化細胞在控制組的數目相對於誘發糖尿病的老鼠是比較多的。更進一步,我們也探討高糖情況下對人類角化細胞的生長、爬行,以及對凝血酶調節素表現的影響。我們發現在高糖的刺激之下會明顯促使凝血酶調節素表現下降,同時也會抑制角化細胞生長及爬行的能力。除此之外,我們利用缺少凝血酶調節素第一個功能區塊的基因轉殖鼠(TMLeD/LeD mice)來研究傷口癒合,相較於TMWt/LeD 老鼠,TMLeD/LeD 老鼠的傷口癒合比較慢,從組織染色得知造成傷口癒合較慢的原因可能是浸潤到傷口中的嗜中性白血球數目增多,而巨噬細胞的數目較少。這些結果建議著凝血酶調節素可能調控角化細胞生長及爬行的能力,並且調控發炎反應,藉此傷口得以完全癒合。
英文摘要 Diabetes mellitus (DM) is characterized as impaired glucose homeostasis. Persistently elevated blood glucose levels in patients with DM lead to complications, including inflammation, endothelial dysfunction, and poor wound healing. Keratinocyte migration and proliferation are required for re-epithelialization of cutaneous wounds and are important to restore barrier function of skin. Thrombomodulin (TM), an anticoagulant glycoprotein, is expressed by endothelial cells and epidermal keratinocytes. However, the expression and function of TM in epithelium remain unclear. In this study, higher expression of TM in the hyperproliferative epithelium (HE) at day 3 and day 5 after full-thickness excisional wound in mice was found, while the expression of TM was postponed at the wound margin in streptozotocin (STZ)-induced diabetic mice. The cell number of proliferating keratinocytes within the HE of control mice was higher than that of STZ-induced mice. Moreover, the effects of high glucose concentration on the expression of TM in keratinocytes and on keratinocyte proliferation and migration in vitro were investigated. High glucose treatment significantly decreased TM expression and inhibited keratinocyte proliferation and migration. In addition, transgenic mice that lack lectin-like domain of TM (TMLeD/LeD mice) exhibited delayed wound healing with higher infiltration of neutrophils and lower infiltration of macrophages when compared with TMWt/LeD mice. Taken together, these results suggest that TM may regulate keratinocyte proliferation and migration as well as inflammation, by which TM may control cutaneous wound healing.
論文目次 Abstract in Chinese 1
Abstract in English 2
Acknowledgement 3
Contents 5
Figure Contents 7
Abbreviation 8
Instruments 9
Reagents and Chemicals 11
Introduction 15
Specific Aim 21
Material and Methods
1.Cell Culture of HaCaT Cells 22
1-1 Cell culture
1-2 Cell counting
1-3 Cell freezing
1-4 Cell thawing
2.General Protein Analysis 24
2-1 Protein sample preparation
2-2 Protein quantification
2-3 Electrophoresis
2-4 Western blotting analysis
3.Immunofluorescent Staining 29
4.Staining of tissue 30
4-1 Paraffin section
4-2 Immunohistochemical Staining (paraffin)
5.Cell proliferation assay (MTT assay) 32
6.Scratch wound healing assay 32
7.Animal : Mice lacking the NH2-terminal lectin-like
domain of TM 33
7-1 Tissue genomic DNA extraction
7-2 Polymerase chain reaction PCR
7-3 Agarose gel electrophoresis
8.Animal : STZ-induced diabetic mice 35
9.Mice skin wound healing 37
9-1 Wound preparation and macroscopic examination
9-2 Wound tissue collection
Results
1.TM is expressed in suprabasal layer of hyperproliferative epithelium during wound healing………………………………39
2.TM expression in suprabasal layer of HE is postponed in streptozotocin (STZ)-induced diabetic mice……………………39
3.STZ-induced diabetic mice show impaired re-epithelialization after wounding due to reduced keratinocyte proliferation………40
4.High glucose levels down-regulate TM expression in human keratinocyte HaCaT cells………………………………………40
5.High glucose concentration inhibits proliferation and
migration of HaCaT cells………………………………41
6.TM lectin-like domain plays a role in anti-inflammation during early stage of wound healing……………………………………42
Conclusion 43
Discussions 44
References 47
Figures 53
Author’s Resume 60
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