臺灣博碩士論文加值系統

matriptase是屬於第二型的嵌膜絲胺酸蛋白酶（type II transmembrane serine protease, TTSP），廣泛表現在人類上皮組織中，其同源抑制蛋白（cognate inhibitor）為肝細胞生長因子活化劑抑制蛋白 （hepatocyte growth factor activator inhibitor 1, HAI-1），而matriptase在活化後會立即與HAI-1結合，藉以調控matriptase的活性。在過去的文獻中發現，人體內matriptase基因產生突變，會導致頭髮較為稀疏且有捲曲的現象，同時皮膚也會出現魚鱗癬的症狀；若將小鼠的matriptase基因剔除，則會導致小鼠的毛髮發育不良，無法突破表皮層生長，皮膚也會有皺縮、乾燥的現象。因此推測matriptase確實與皮膚的分化以及毛囊的正常發育有一定的相關。皮膚分化與毛囊發育過程中，目前已知有上皮幹細胞（keratinocyte stem cell）參與其中，進而調控正常的增生分化；除此之外還有些重要的訊號傳遞路徑與增生分化有相關，例如肝細胞生長因子 (hepatocyte growth factor, HGF) 與其受器c-Met。同時，傷口癒合的過程中上皮的增生與分化也是很重要的一個過程。因此本研究藉由取得正常皮膚組織與慢性傷口組織，來探討matriptase在兩者間表現的差異，最後藉由酸處理ex vivo實驗來討論matriptase是否在傷口癒合的過程中有參與。結果可以發現，(1) 在正常皮膚組織以及慢性傷口組織中，matriptase的分布皆是由表皮層的基底層向上遞減，HAI-1的分布則是位於整個表皮層。(2) 活化態matriptase在正常皮膚組織中的表皮層以及毛囊都會與c-Met、K15表現在共同的位置上。但在慢性傷口組織中，活化態matriptase表現量極低，且K15完全不表現。(3) 以酸處理HaCaT細胞、正常皮膚組織以及慢性傷口都可以看到matriptase的活化。根據實驗結果可知活化的matriptase在正常皮膚組織中與c-Met、K15的表現位置非常類似，可能有某種交互作用，同時matriptase在酸性環境中的活化，可以推測或許有參與上皮再生的過程。

Matriptase, a member of the type II transmembrane serine proteases, is widely expressed in virtually all epithelia. After activation, matriptase is bound directly by its cognate inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Matriptase knock-out mice have shown abnormal development of epidermis and hair follicle. The hair of one ARIH (autosomal recessive ichthyosis and hypotrichosis) patient, whose matriptase gene is mutated, appeared curly, sparse and dry, and ichthyotic skin was observed. Based on these finding, it is suggested that matriptase plays an important role in epidermis differentiation and hair follicle development. Besides matriptase, HGF, c-Met, and stem cells could be involved in epidermis differentiation and hair follicle development. In this study, we utilize immunohistochemistry to examine the expression of matriptase in normal and chronic wound human skin tissue. We also use mild acidic condition to mimic the wound healing process from alkaline to acidic, tried to understand whether matriptase is involved in wound healing or not. According to our results, we have some conclusions. First, in normal human skin and chronic wound tissue, HAI-1 is distributed evenly in epidermis, however matriptase is highly expressed in the basal layer and decreased to none alone with the terminal differentiation in the spinous layer. Second, activated matriptase, c-Met and stem cell marker, K15, are co-localized in the basal layer of normal human skin and the outer root sheath of hair follicle. But in chronic wound, activated matriptase and c-Met are rarely observed in basal layer, and K15 totally can’t be detected in basal layer. Third, after treating with mild acidic buffer, HaCaT cells, normal skin tissue, and chronic wound exhibit highly activated matriptase. These results suggest that the distribution of activated matriptase is similar to c-Met and K15. They may have strong correlations in regulating epidermis differentiation and hair follicle development. Besides, matriptase can be activated in acidic condition, it may involve in some reactions during wound healing from alkaline to acidic.

表目錄 IV
圖目錄 V
縮寫表 VI
摘要 VII
Abstract IX
第一章　緒論 1-24
第一節　Matriptase 1
一、Matriptase的發現 1
二、Matriptase的結構 2
三、Matriptase的分布與生理功能 3
第二節　HAI-1 5
一、HAI-1的結構 5
二、HAI-1的功能 6
第三節　Matriptase與HAI-1 7
一、Matriptase活化的過程 7
二、可能活化Matriptase的機制 8
三、Matriptase的下游反應 9
四、Matriptase與HAI-1的平衡 11
第四節　皮膚 12
一、皮膚的基本構造 12
二、表皮層的結構 13
三、皮膚的附屬器官（skin appendages） 15
四、上皮幹細胞（keratinocyte stem cell） 18
第五節　慢性傷口（chronic wound） 20
一、慢性傷口的定義 20
二、糖尿病足潰瘍 20
三、傷口癒合（wound healing） 21
四、pH值對傷口癒合的影響 23
第六節　實驗目的 24
第二章　材料與方法 25-34
第一節　實驗材料 25
ㄧ、藥品試劑 25
二、套裝實驗組 26
三、耗材 26
四、抗體 26
五、實驗儀器 27
六、檢體的取得 27
第二節　緩衝液（Buffer）與SDS-PAGE之製備 28
一、Lysis Buffer 28
二、5X loading buffer 28
三、Running buffer（pH 8.3） 28
四、Transfer buffer（pH 8.3） 29
五、SDS-PAGE（8.5％） 29
六、Sodium Phosphate buffer 29
第三節　實驗方法 30
一、檢體處理 30
二、冷凍切片 30
三、免疫組織化學染色（Immunohistochemistry） 31
四、蘇木紫與伊紅染色（Hematoxylin & Eosin stain） 32
五、體外實驗（ex vivo） 32
六、蛋白質濃度之測定 32
七、西方墨點法（western blot）樣本之製備 33
八、西方墨點法（western blot analysis） 33
九、濃縮（concentration） 34
第三章　結果 35-42
第一節　Matriptase及HAI-1在正常皮膚組織中的表現 35
一、表皮 35
二、毛囊 35
三、皮脂腺 36
四、汗腺 36
第二節　活化態Matriptase、c-Met及K15在正常皮膚組織中的表現 37
一、活化態Matriptase在正常皮膚中的分布 37
二、c-Met 在正常皮膚中的表現 38
三、K15 在正常皮膚中的表現 38
四、活化態Matriptase 與 c-Met、K15 的共同表現 39
第三節　Matriptase與慢性傷口 40
一、慢性傷口中Matriptase與HAI-1的表現 40
二、慢性傷口中活化態Matriptase、c-Met與K15的表現 40
三、慢性傷口的pH值變化對Matriptase表現的影響 41
第四章　討論 43-51
第一節　Matriptase在正常皮膚中可能扮演的角色 43
一、表皮分化 43
二、毛囊發育 44
第二節　正常皮膚組織與慢性傷口組織的表現差異 45
第三節　Matriptase可能參與傷口癒合的過程 47
第四節　Matriptase與慢性傷口的發炎反應 49
第五節　結論 50
第六節　未來實驗方向 51
第五章　參考文獻 52

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