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系統識別號 U0026-0812200914201729
論文名稱(中文) 凝血酶調節素缺失對皮膚上皮細胞分化與傷口癒合之影響
論文名稱(英文) Epithelium-specific deletion of thrombomodulin disrupts the epidermal differentiation and cutaneous wound healing processes
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) of Biochemistry and Molecular Biology
學年度 96
學期 2
出版年 97
研究生(中文) 吳雨庭
研究生(英文) Yu-Tin Wu
電子信箱 alwaysgirl@yahoo.com.tw
學號 s1695112
學位類別 碩士
語文別 英文
論文頁數 93頁
口試委員 口試委員-蔡曜聲
口試委員-林淑華
指導教授-施桂月
口試委員-吳華林
中文關鍵字 初代角質細胞  凝血酶調節素  傷口癒合  皮膚  上皮細胞分化 
英文關鍵字 skin  keratinocyte  primary culture  wound healing  differentiation  epidermal  Thrombomodulin 
學科別分類
中文摘要 上皮組織是由一連串分層的上皮細胞所組成,在皮膚上形成一層保護膜。凝血酶調節素(Thrombomodulin,簡稱TM)是一個type I 穿膜醣蛋白,最早被發現在血管內皮細胞,扮演著抗凝血的角色。凝血酶調節素在結構上被區分為五個功能區,第一個部分為似lectin的功能區 (TMD1),第二個部份是由六個似表皮生長因子所組成的功能區(TMD2),第三個部分是富含絲胺酸/息寧胺酸胺基酸區域(TMD3),接著第四和第五個功能區分別為穿膜功能區(TMD4)及細胞質功能區(TMD5)。凝血酶調節素已被發現位於上皮的棘狀層(spinous layer)及受傷後的新生上皮細胞中,但有關凝血酶調節素的真正功能目前仍未清楚。因此,我們欲研究凝血酶調節素在皮膚上及皮膚受傷過程中可能扮演的生理意義。首先我們建立了角質細胞缺失凝血酶調節素的動物模式-TMLox/Lox‧Cre mice。並由小鼠尾巴上皮分離出初代角質細胞(keratinocytes)做為研究凝血酶調節素在分化(differentiation)和爬行(migration)之功能分析。將培養液中鈣離子濃度由低濃度(0.06 mM)置換成高濃度(1.2 mM)會促使初代角質細胞分化。凝血酶調節素則會伴隨著分化現象發生而表現。另一方面,我們亦發現缺失凝血酶調節素的初代角質細胞即使給予高濃度的鈣亦無法趨向分化的型態,且鈣黏著素E(E-Cadherin)不會位於細胞間隙間(cell-cell junction)。缺失凝血酶調節素的TMLox/Lox‧Cre老鼠其皮膚細胞核呈現較圓的狀態,上皮細胞分化標記蛋白filaggrin以及loricrin表現量降低。更進一步,在刮傷癒合分析(scratch wound healing assay)中缺乏凝血酶調節素的初代角質細胞與對照組相比其癒合速度較慢。在角質細胞缺乏凝血酶調節素的TMLox/Lox‧Cre 老鼠中進行傷口癒合分析,以組織學的觀察可發現TMLox/Lox‧Cre老鼠的新生上皮層數較少,且傷口癒合速度也比較慢;其血管新生(neovasculization)跟肌纖維母細胞(myofibroblast)的增生以及傷口收縮跟對照組比較起來也有減少的現象。我們亦證實當外加重組凝血酶調節素 TMD23可顯著改善 TMLox/Lox‧Cre老鼠傷口癒合的情況。由上結果我們證實凝血酶調節素參與了皮膚上皮細胞分化以及傷口癒合的過程。
英文摘要 Epidermis is a stratified squamous epithelium forming the protective covering of the skin. Thrombomodulin (TM), a type I transmembrane glycoprotein originally identified in vascular endothelial cells and is well-characterized as an anticoagulant factor. TM has five domain structures comprising a lectin-like domain (TMD1), six tandem epidermal growth factor-like domains (TMD2), a serine/threonine-rich domain (TMD3), a transmembrane domain (TMD4), and an intracellular domain (TMD5). TM is highly expressed in spinous layer of epidermis and stratifying keratinocytes within the neoepidermis in wounds. However, the functions of TM in skin have not been well-studied. Investigated the role of TM in the skin by creating a mouse model with keratinocyte-restricted deletion of TM, the TMLox/Lox‧Cre mice. Primary keratinocytes isolated from the tail skin of the mice were prepared for the study of TM functions in cell differentiation and migration. Cells maintained in the low-calcium medium (0.06 mM) were stratified and differentiated by culturing in the high-calcium medium (1.2 mM). TM was not expressed in the undifferentiated keratinocytes. On the other hand, TM-null keratinocytes kept in high-calcium medium appeared as rounded shape, which is one characteristic of undifferentiated cells, indicating that they did not undergo differentiation. Immunohistochemical analysis showed that the nucleus of the TM-null epidermis granular layer appeared in square shape. Furthermore, the expression of differentiation markers, loricrin and filaggrin, were decreaed in TMLox/Lox‧Cre mice epidermis. E-cadherin, an essential molecule in promoting cell differentiation, was not distributed at the cell junction of the TM-null keratinocytes. We further identified that the undifferentiated TM-null keratinocytes was slower in migration by in vitro scratch wound healing assay consitaint with this. In vivo cutaneous wound healing assay showed that the wound closure processes were delayed in TMLox/Lox‧Cre mice. The histological examinations demonstrated that the hyperproliferative epithelium was thinner and that the vascularization, myofibroblast formation, and wound contraction were also lower in TMLox/Lox‧Cre mice than those of wild type mice. Importantly, treatment of recombinant mouse TMD23 can significantly promote wound closure and recover wound contraction in the TM Lox/Lox‧Cre mice. Therefore, our results suggest that TM is involved in cell differentiation and wound healing processes of the skin.
論文目次 Abstract in Chinese ......................................1
Abstract in English ......................................2
Acknowledgement ..........................................4
Contents..................................................5
Figure Contents...........................................7
Appendix Contents.........................................8
Abbreviation..............................................9
Reagents.................................................10
Introduction.............................................14
1.Thrombomodulin.
1-1.Structure of TM......................................14
1-2.Distribution and Function of TM .....................14
1-3.TM expression in epidermis of the skin...............15
2.Skin structure and epidermal differentiation
2-1. Skin structure......................................16
2-2.Differentiation process of epidermis.................17
3.Skin wound healing process
3-1.Importance of wound healing..........................18
3-2.Wound healing stages.................................19
3-3.Therapy of wound healing.............................20
Specific Aim.............................................21
Material and Method......................................22
1.Expression of recombinant mouse TMD23 protein..........22
1-1.Yeast Culture and Induction..........................22
1-2.Protein Purification.................................23
1-3.SDS-Polyacrylamide Gel Electrophoresis...............24
1-4.Western Blot.........................................26
2.Purification of mTMD23 polyclonal antidody.............28
2-1.Immobilization of mTMD23 protein to CNBr-ativated SepharoseTM..............................................28
2-2.Purification of mTMD23 polyclonal antibody...........30
2-3.Enzyme-linked immunosorbent assays (ELISA)...........30
2-4.Silver Stain.........................................32
3.Animal: keratinocyte conditional knockout of TM .......33
3-1.Tissue genomic DNA extraction .......................33
3-2.PCR .................................................34
3-3.Agarose gel electrophoresis..........................36
4.Adult murine keratinocyte primary culture .............36
4-1.Isolation of basal keratinocyte......................36
4-2.Primary keratinocyte subculture......................38
5.Cell culture of HaCaT cells............................39
5-1.Cell culture.........................................39
5-2.Cryopreservation of cultured cells ..................40
5-3.Retrieval of cells from frozen storage...............41
6.Spreading assay........................................42
7.Immunofluorescent staining.............................42
8.Mouse skin wound healing and mTMD23 treatment..........44
9.Microtone staining of tissue ..........................45
9-1.Preparation of the slides............................45
9-2.Paraffin section ....................................45
9-3.Heamatoxylin and Eosin Stain (parafin)...............46
9-4. Heamatoxylin and Eosin Stain (frozen)...............48
9-5. Masson trichrome stain .............................49
9-6. Immunohistochemical staining (frozen)...............51
Results .................................................53
Discussion ..............................................60
References...............................................67
Figures .................................................72
Appendices ..............................................87
Resume...................................................93
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