臺灣博碩士論文加值系統

當成年的哺乳類動物出現傷口，通常會形成疤痕並且癒合。由過量膠原蛋白，纖維蛋白組成的疤痕雖然可以癒合傷口，卻會阻礙組織當中細胞的訊息傳遞與再生能力，並且失去正常皮膚組織具備的功能。這項研究的目的是為了瞭解在傷口癒合的期間，受到Msx2組成不同蛋白複合體來進行調控，並且在傷口中央(再生毛囊)與傷口邊緣(形成疤痕)有所不同的組織再生能力。先前的研究顯示在面積較大的傷口中間會有毛髮的再生，這個現象也被稱作「傷口誘導毛囊再生」(Wound-Induced Hair follicle Neogenesis, WIHN)。有趣的是，先前研究發現Msx2基因, muscle segment homeobox 2以及它的蛋白質(轉錄因子)Msx2在小鼠的傷口誘導毛囊再生(WIHN)模式中也扮演重要的角色：在一個移除上皮層及真皮層的傷口中，Msx2在癒合早期表現在傷口周圍，然而在後期Msx2表現在傷口中央，也就是毛囊再生的地方。Msx2在時間與空間上的表現差異可以對應到傷口誘導毛囊再生的過程，這樣的現象引發我們進一步去研究Msx2在傷口誘導毛囊再生中促進細胞重新編碼的調控機制，以及Msx2是否可以調控細胞的狀態，又或者是否受到表觀遺傳學的影響而進行細胞再程序化進而提升組織再生潛能。我們假設Msx2會引導不同訊號傳遞路徑之蛋白質或表觀遺傳學的效應物形成複合體，並且在傷口周圍及傷口中央藉由調控不同細胞狀態而調控組織再生的能力與效果。
我們發現在傷口周圍及中央有兩種不同調控情形。在傷口周圍，訊號路徑上的效應物，例如：β-catenin, LEF1和SHH，及表觀遺傳學調控因子，例如：HDAC1, HDAC2和EZH2，有較高的表現。這些調控因子會和Msx2交互作用而構成不同的蛋白質複合體進而抑制組織再生。再者，由於表觀遺傳的抑制作用，Msx2啟動子上的LEF/TCF 及Smad結合位都是關閉的，使得轉錄作用在傷口周圍較難以進行。另一方面，在傷口中央有另一套調控機制，除了拓撲異構酶topoisomerase II-β (TOPIIβ)，訊號路徑效應物及表觀遺傳學調控因子大多有較低的表現量。拓撲異構酶II-β (TOPIIβ)可以藉由解開雙股螺旋的構造進而改變染色質纏繞的情形，並且促進基因表現。在傷口中央，大多數的抑制蛋白和拓撲異構酶topoisomerase II-β (TOPIIβ)會脫離Msx2來停止抑制作用及開始細胞再程序化進而誘導組織再生。另外，Msx2啟動子上的LEF/TCF結合位受到TOPIIβ的調控以及表觀遺傳在組蛋白H3K9和H3K27上的修飾而展開，所以利於Msx2基因表現。而我們也發現LEF1的確會與Msx2啟動子上的LEF/TCF結合位結合，而β-catenin也表現在傷口中央的細胞核中，這表示WNT訊號路徑調控Msx2的表現並促進傷口誘導毛髮再生的現象。總而言之，我們發現了Msx2引導之兩種結合訊號傳遞路徑與表觀遺傳學的不同調控機制，在傷口中央進行細胞再程序化並促進組織再生。藉由以上知識，我們可以更加了解如何藉由內源性調控來促進正常組織的再生進而幫助有嚴重傷口的病患。

In adult mammals, wounds usually heal by forming scars. Scar consists of excessive disorganized collagen, fibroblasts and myofibroblasts. This disorganized structure can block off cell communication preventing regeneration. Additionally, this process loses the functions of normal healthy tissue. The purpose of this study is to understand the different ability to regenerate at wound margin (non-regenerative) versus wound center (regenerative) which might be regulated by different complexes guided by Msx2 during wound healing. Previous studies showed there is hair neogenesis in a large wounds in mammals which is known as “Wound Induced Hair follicle Neogenesis” (WIHN) (Chuong 2007, Ito, Yang et al. 2007). Interestingly, Msx2, muscle segment homeobox 2, has been proved to play a role during skin regeneration in axolotl and mice. In mice, Msx2 is expressed at the wound margin at an early stage, while it is expressed at the wound center at a late stage co-localizing where hair neogenesis occurred during full-thickness wound healing. The spatio-temporal expression change of Msx2 piqued our curiosity as to the functional role of Msx2 and if the different cell states between wound margin and center are regulated by Msx2. The underlying mechanism may also involve epigenetic modulation to cellular reprogram. We hypothesize Msx2 directs differential protein complexes, including pathways effectors and epigenetic regulators, to modulate regeneration via different cell states at the wound margin/non-regenerative versus the wound center/regenerative.
Our data suggested two regulatory profiles at the wound margin and the wound center. At the wound margin, signaling effectors like β-catenin, LEF1, SHH and epigenetic regulators like, HDAC1, HDAC2 and EZH2 show higher expressions. These regulators interact with Msx2 to build up different protein complexes to inhibit the tissue regeneration. Moreover, the Msx2 promoter is close at both LEF/TCF and Smad binding elements due to the epigenetic repression of histone modifications. On the contrary, at the wound center, signaling effectors and epigenetic regulators show lower expressions except for TOP2b which is response for remodeling the chromatin packaging and allow genes to express. Most of the repressor proteins and TOP2b release from Msx2 to stop the inhibition and start cellular reprogramming at the wound center. In addition, the Msx2 promoter is open at the LEF/TCF binding element with the aids of acetylation on H3K9 and H3K27 and TOP2b. LEF1 bind to the LEF/TCF binding element on Msx2 promoter which suggests that WNT pathway regulates the expression of Msx2 and promote hair follicle neogenesis during wound healing at the wound center. Taken together, Msx2 guides two regulatory profiles of genetic and epigenetic regulations at the wound margin and the wound center to carry out different ability to regenerate.

ABSTRACT I
中文摘要 III
致謝 V
CONTENT VI
ABBREVIATIONS VII
INTRODUCTION 1
Skin biology 1
Hair follicle morphogenesis and Hair cycle 2
Stem cell and tissue competency (repair/adult vs regeneration/fetal) 3
Wound Induced Hair follicle Neogenesis (WIHN) 5
The expression of Msx2 during WIHN 5
Cellular reprogramming of hair follicle neogenesis 6
Epigenetic and endogenous reprogramming 7
Significance 10
Hypothesis 10
Specific aims 10
METERIALS AND METHODS 11
Mice 11
Wound induced hair follicle neogenesis (WIHN) assay 11
Whole mount tissue staining 11
Immunohistochemistry (IHC) staining 12
Mouse skin tissue protein extraction 12
Western blot 13
Co-immunoprecipitation (co-IP) assay 13
Mouse skin tissue protein-DNA complex extraction 14
Chromatin immunoprecipitation (ChIP) assay 14
RESULTS 15
Aim1: To characterize the differential expression pattern of genetic and epigenetic markers associated with wound regeneration at late stage during WIHN at wound center. 15
Epigenetic enzymes and modification markers expressed at wounded center versus margin during the wound healing process. 15
Epigenetic modification markers showed different distribution patterns at wound margin versus wound center during healing process. 15
Genetic regulators showed different expression levels at wound margin (non-regenerative) versus wound center (regenerative) during healing process. 16
Epigenetic enzymes showed different expression levels at wound margin (non-regenerative) versus wound center (regenerative) during the healing process. 17
Aim2: To identify protein complexes associated with Msx2 of regeneration at the wound center and repair at the wound margin. 24
Msx2 associates with multiple genetic and epigenetic regulators during WIHN. 24
Aim3: To identify the upstream signals regulating Msx2 at wound margin versus wound center during WIHN. 28
Msx2 promoter is bound by LEF1 at the wound center during WIHN. 28
CONCLUSION 31
DISCUSSION 33
SUPPLEMENTARY 41
REFERENCE 45

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