蟹足腫是一種纖維化的皮膚疾病，蟹足腫病人在受傷過後過多細胞外基質累積在皮膚真皮層。我們實驗室發現微囊蛋白1 (Caveolin-1) 在蟹足腫纖維母細胞表現低落並促進細胞分泌更多細胞外基質、細胞軟化、外界機械力感測能力喪失等等狀。現在愈來愈多證據指出蟹足腫傷疤形成與機械力的刺激有關係，蟹足腫疤痕只出現於人體高張力硬度的區位，也因此我們認為蟹足腫疾病不只是個體基因差異所致，表觀遺傳學也著實地調控著蟹足腫傷疤的生成。根據Tanya J. Shaw教授的研究，我們希望能瞭解去乙醯蛋白2 (HDAC2)可能在蟹足腫生成所扮演的角色。首先我們使用去乙醯蛋白酶抑制劑-Trichostatin A (TSA) 會增加正常與蟹足腫纖維母細胞的微囊蛋白1表現量增加。蟹足腫組織有比較多的去乙醯蛋白2表現，但是在細胞實驗卻看不出顯著的差異。當細胞培養在軟硬不同的材質上時，正常與蟹足腫纖維母細胞的去乙醯蛋白2會隨著機械力增加而表現增加。可是使用小分子干扰核糖核酸(siRNA)抑制去乙醯蛋白表現時並不影響微囊蛋白1的表現。並且正常的傷疤與肥厚性疤痕也都有明顯有著去乙醯蛋白2的表現。接著我們查詢了高通量基因表達數據庫(GEO No.: GSE90051)。7位日本蟹足腫病人以蟹足腫組織與自身正常組織做比較，發現了DNA甲基轉移酶3A與3B(DNMT3A and DNMT3B)在蟹足腫組織表現增加，而不同的去乙醯蛋白酶並無明顯趨勢。於是我們使用了DNA甲基轉移酶抑制劑(5-aza-2’- deoxycytidine)可以促進蟹足腫纖維母細胞的微囊蛋白1 增加。在蟹足腫組織之中DNA甲基轉移酶1、3A與3B都有表現增加的況但是在細胞實驗中並無明顯差異。將細胞培養在不同軟硬材質時DNA甲基轉移酶1、3A與3B也無明顯差異。而當我們做原代培養(primary culture)，我們發現蟹足腫細胞可以突破組織在不同軟硬度材料上生長，而正常細胞只能在硬材質上突破組織生長，去分析細胞發現DNA甲基轉移酶1與3B在細胞突破組織於硬度材有比較高的表現。除此之外為了更確認DNA甲基轉移酶在蟹足腫所扮演的角色，我們分析了正常與蟹足腫組織的囊蛋白1基因上CpG島岸(CGI shore)甲基化程度。蟹足腫組織的微囊蛋白1基因上的CpG島岸甲基化程度高於正常組織。當蟹足腫纖維母細胞受到甲基轉移酶刺激之後CpG島岸甲基化程度明顯的下降。但是當使用小分子干擾核糖核酸抑制甲基轉移酶時無法使微囊蛋白1增加，取而代之的是能抑制正常和蟹足腫纖維母細胞與骨頭形成有關的轉錄因子-RUNX2表現下降。最後我們使用乙型轉化生長因子1 (TGF-β1)抑制微囊蛋白1，但似乎不受到甲基轉移酶所調控。綜合上面所述蟹足腫傷疤是一個多基因控制的疾病，其受到不同表觀遺傳學分子的調控，我們推測去乙醯蛋白2在正常傷口癒合中就會產生，而甲基轉移酶則綜合性地調控著微囊蛋白1以此促進蟹足腫的生成。

Keloid scar is a fibrotic skin disease resulting from the overproduction of extracellular matrix (ECM) deposited in the dermis. We previously found that the absence of caveolin-1 (CAV1) plays an essential role in keloid pathogenesis, such as ECM production, cell softening, and their rigidity-sensing ability. In the present thesis, we aim to understand how CAV1 expression became absent or largely decreased in keloid fibroblasts. More evidence on the abnormal mechanical force was proven to be associated with keloid formation. Keloids tend to occur in high tension area instead of low tension area on the human body. Therefore, we consider epigenetic regulation, in addition to genetic mutations, participates in the pathogenesis of keloid. First, we investigated HDAC2 function according to Fitzgerald O'Connor, E.J., et al. The HDAC inhibitor, TSA, elevated CAV1 in normal and keloid fibroblasts. Keloid tissues exhibited higher HDAC2 level than normal tissues. However, HDAC2 levels were not significantly different in the keloid fibroblasts and normal fibroblasts cultured in the dishes. We also found HDAC2 was elevated when normal and keloid fibroblasts were seeded on substrates of different stiffness. However, knockdown of HDAC2 did not alter CAV1 expression in normal or keloid fibroblasts. Normal scar and hypertrophic scar also exhibited HDAC2 expression in both the epidermis and dermis. Therefore, we believe HDAC2 may express during the wound healing process, and the expression level is not specific or relevant to keloid formation. Next, we analyzed the published microarray data of keloids versus adjacent normal skin from 7 Japanese patients (GEO No.: GSE90051); HDACs levels were different in keloid tissues of 7 patients, while DNMT3A and DNMT3B were significantly increased in keloid tissues compared to normal tissues. It seemed that DNA methyltransferase was more critical than HDACs in keloid pathogenesis. We found that 5-aza-2’-deoxycytidine, a DNMTs inhibitor, restored CAV1 expression in only keloid fibroblasts. Although DNMT1, 3A, and 3B levels were higher in keloid tissues than normal tissues, the expression of DNMTs seemed not significantly different between normal and keloid fibroblasts in vitro. However, DNMT3B levels were not different in either normal or keloid fibroblasts after mechanical stimulation. Primary culture on substrates with different stiffness showed higher outgrowth potential for keloid fibroblasts then normal fibroblasts. Both DNMT1 and 3B were elevated in the cells seeded in stiffer substrates. To confirm the DNMTs' role in CAV1 expression in keloid pathogenesis, CAV1 CpG island shore methylation level was tested in both normal and keloid tissues and fibroblasts. Keloid tissues exhibited higher methylation level in the CAV1 CGI shore. After 5-aza-2’- deoxycytidine treatment, keloid fibroblasts showed decreased methylation level in CAV1 CGI shore. Knocking down DNMT 3B blocked RUNX2 instead of the CAV1 expression in fibroblasts. TGF-β1 inhibited CAV1 without affecting DNMTs expression. In conclusion, different epigenetic regulation may be involved in keloid development among different patients. We speculated that HDAC2 was produced in a normal wound healing process, while DNMTs isoforms might regulate CAV1 expression in keloid pathogenesis.

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