臺灣博碩士論文加值系統

第二型糖尿病長期的病人經常會伴隨著末稍血液循環不良以及血管和神經的萎縮，使傷口處於缺氧狀態，也讓免疫反應無法有效作用，最終轉變成慢性傷口。實驗室先前的結果顯示，matriptase和prostasin這兩種絲胺酸蛋白酶在慢性傷口的癒合過程中皆扮演重要的角色。因此，本次我們想要比較並探討糖尿病足部與非足部的慢性傷口癒合良好或不佳的表皮組織中 matriptase、prostasin及其抑制蛋白HAI-1和HAI-2會有甚麼變化。
在此次研究中我們發現 （1）與糖尿病足相比，在糖尿病非足部的組別中，matriptase和prostasin的表現範圍都會跟normal組別極其類似，不過HAI-1的表現強度則下降許多。 （2）在傷口癒合良好的組別中，雖然matriptase和prostasin的表現有與傷口癒合不佳的組別有明顯的差異，但是matriptase的表現範圍依舊沒有回升跟normal組別一樣，以及prostasin的表現範圍也不能完全收斂回顆粒層中。表示雖然臨床上最終慢性傷口癒合良好，但在清創過程中取得的組織狀況依舊是不好的。 （3）糖尿病的慢性傷口上，約45.4%的組織中有活化態matriptase表現在上方棘狀層或顆粒層的細胞膜上，同時也發現，在此類型組織中相似位置上的HAI-1明顯地表現強度較強，而且高醣基化HAI-2 （DC16）也出現在相似位置的細胞膜上。並且在不朽化的人類角質細胞株HaCaT中，利用免疫沉澱法，驗證了活化態matriptase和高醣基化HAI-2 （DC16）會形成100 kDa的複合體。因此可以推論：高醣基化HAI-2 （DC16）可以幫助HAI-1而表現在相似位置的細胞膜上，用來抑制過多的活化態matriptase。 （4）在同一次手術清創取下糖尿病慢性傷口的大組織中，不管傷口癒合良好或不佳，從較健康處一直延續至靠近傷口處時，發現matriptase的漸層表現範圍會隨之降低，並在靠近傷口處，活化態的prostasin會明顯的向下延伸表現在棘狀層中。因此可以推論，在傷口附近有著某種原因導致這兩種絲胺酸蛋白酶及其抑制蛋白有著截然不同的表現。 （5）利用HaCaT細胞株，提高培養基中鈣離子濃度或者提升細胞密度，促使細胞走向終端分化後，除了細胞群聚的現象會增加外，也發現 matriptase 、prostasin、 HAI-1及高醣基化HAI-2 （DC16）也會隨著分化而表現增加。因此可以推論：終端分化除了影響prostasin的表現外，還會影響其他蛋白質的表現。
本篇論文中，我們觀察出在不同的糖尿病潰瘍程度中，matriptase、prostasin、HAI-1以及HAI-2的表現會有所不同，表示這些蛋白質可能與傷口癒合的不同時期有密切關係。因此，我們期盼可以找出針對慢性傷口癒合的相關策略，以提供臨床上可能的治療方法。

The type II diabetic patients have poor peripheral blood circulation and vascular and nerve atrophy, which induce hypoxia condition and non-effective immune response. These effects may trigger the formation of chronic wound. Previous results have shown matriptase and prostasin are important in chronic wound healing. Therefore, we would like to learn the expression of matriptase and prostasin in different clinical outcome of chronic diabetic ulcers and compare the differences between foot and non-foot area.
At first, compared with DM foot ulcer, the expression extent of matriptase and prostasin in DM non-foot ulcer was similar with normal skin, but the expression intensity of HAI-1 was still lower like DM foot ulcer. Secondly, although the expression of matriptase and prostasin are different between better outcome and worse outcome of diabetic ulcer, however the expression of matriptase could not extend to normal skin, and the expression extent of prostasin also could not withdrawal to the upper spinous layers. Thus, we suggested that the tissues after debridement, in the better outcome of DM ulcer, are still in bad condition. Thirdly, we discovered 45.4 percent DM ulcers have shown activated matriptase on the cell membrane in the upper spinous and granular layers, which is similar with the activated prostasin expression. At the same location, mostly, the expression intensity of HAI-1 was stronger, and highly glycosylated HAI-2 （DC16）also was detected. Further, in immortalized human keratinocytes cell line （HaCaT）, we found a 100 kDa complex activated matriptase and highly glycosylated HAI-2 by using immunoprecipitation. Therefore, we suggested that highly glycosylated HAI-2 was expressed on the cell membrane in the upper spinous and granular layers because they can help HAI-1 to inhibit more activated matriptase. Fourthly, the whole tissues of DM ulcer from the same debridement could expand from the healthy side to the wound side. We found that the expression extent of matriptase at the wound side was lower than the healthy side. At the same time, activated prostasin was spread out more from granular layers to the spinous layers at the wound side. Thus, we recommend that there were some reasons to make the dissimilar distribution of matriptase, prostasin, and their inhibitors between healthy side and wound side. Finally, we used HaCaT cells to study the expression of prostasin under terminal differentiation. We tried to mimic the terminal differentiation condition by increasing calcium concentration or increase cell density. In addition to more cell clustering, we found the expression of matriptase, prostasin, HAI-1, and HAI-2 were increased under differentiation. Hence, we suggested that the differentiation of human keratinocytes did not only influence prostasin, but also induce the expression of other proteins.
In this thesis, we observed the expression of matriptase, prostasin, HAI-1 and HAI-2, and concluded that these proteins are closely related in wound healing at different stages. Therefore, we can find the possible strategy for chronic wound healing, and this information will provide clinical treatment to accelerate chronic wound healing.

目錄
表目錄 VII
圖目錄 VIII
縮寫表 XIV
中文摘要 XV
Abstract XVII
第一章 緒論 1
第一節 皮膚 1
壹、皮膚組織結構 1
貳、表皮層之分層 3
參、皮膚的附屬器官 （skin appendages） 5
肆、皮膚幹細胞 （skin stem cell） 7
伍、表皮層的終端分化 （terminal differentiation） 8
第二節 慢性傷口 （chronic wound）的演進 8
壹、傷口癒合 （wound healing） 8
貳、急性傷口 （acute wound） 11
參、慢性傷口 （chronic wound） 11
第三節 matriptase 14
壹、蛋白質水解酶 （protease） 14
貳、絲胺酸蛋白酶 （serine protease） 14
參、matriptase 的發現 15
肆、matriptase 的結構 16
伍、matriptase 的分佈與生理功能 17
陸、matriptase 的病理角色 18
第四節 prostasin 19
壹、prostasin 的發現 19
貳、prostasin 的結構與活化 19
參、prostasin 的分佈位置及生理功能 20
第五節 Hepatocyte growth factor activator inhibitor-1 （HAI-1） 20
壹、HAI-1的發現 21
貳、HAI-1的結構 21
參、HAI-1的分佈與功能 22
第六節 Hepatocyte growth factor activator inhibitor-2 （HAI-2） 22
壹、HAI-2的發現 22
貳、HAI-2的結構 23
參、HAI-2的分佈與功能 23
第七節 matriptase與HAI-1及HAI-2 24
壹、matriptase的活化 24
貳、matriptase可能的活化機制 25
參、matriptase的下游反應 26
肆、matriptase與HAI-1的平衡 27
伍、matriptase與HAI-2的平衡 28
第八節 實驗室的研究成果 28
第九節 研究目的 29
第二章 實驗材料與方法 30
第一節 實驗材料 30
壹、試劑 30
貳、套裝實驗組 32
參、抗體 32
肆、實驗耗材 34
伍、實驗儀器 34
陸、實驗道德倫理聲明 （ethics statement） 35
第二節 實驗方法 35
壹、檢體處理 35
貳、冷凍切片 （Frozen section） 35
參、免疫組織化學染色 （Immunohistochemistry, IHC） 36
肆、蘇木紫與伊紅染色 （Hematoxylin & Eosin stain, HE stain） 38
伍、封片與組織拍照 38
陸、人類皮膚組織萃取液製備過程 38
柒、細胞培養 （Cell culture） 39
捌、細胞酸處理 （Treatment of acidic buffer） 40
玖、免疫沉澱法 （Immunoprecipitation, IP） 40
拾、西方墨點轉漬法 （Western blotting） 41
拾壹、免疫組織化學染色判讀 （interpretation of IHC） 43
拾貳、統計分析 （Statistical analysis） 43
第三章 實驗結果 45
第一節 比較糖尿病足與糖尿病潰瘍在一次清創中傷口癒合良好或不佳的蛋白質表現差異 45
壹、total matriptase （M24）的表現差異 45
貳、活化態matriptase （M69）的表現差異 46
參、HAI-1 （M19）的表現差異 46
肆、高醣基化HAI-2 （DC16）的表現差異 47
伍、total prostasin （YL11）以及活化態prostasin （YL89）的表現差異 47
陸、皮膚幹細胞標記蛋白K15的表現差異 48
第二節 比較在糖尿病足多次清創前後傷口癒合良好或不佳的蛋白質表現差異 48
壹、total matriptase （M24）的表現差異 49
貳、活化態matriptase （M69）的表現差異 49
參、HAI-1 （M19）的表現差異 49
肆、高醣基化HAI-2 （DC16）的表現差異 50
伍、total prostasin （YL11）以及活化態prostasin （YL89）的表現差異 50
陸、皮膚幹細胞標記蛋白K15的表現差異 51
第三節 比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的蛋白質表現差異 51
壹、total matriptase （M24）的表現差異 52
貳、活化態matriptase （M69）的表現差異 52
參、HAI-1 （M19）的表現差異 52
肆、高醣基化HAI-2 （DC16）的表現差異 53
伍、total prostasin （YL11）以及活化態prostasin （YL89）的表現差異 53
陸、皮膚幹細胞標記蛋白K15的表現差異 54
第四節 比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的蛋白質表現差異 54
壹、total matriptase （M24）的表現差異 54
貳、活化態matriptase （M69）的表現差異 55
參、HAI-1 （M19）的表現差異 55
肆、高醣基化HAI-2 （DC16）的表現差異 55
伍、total prostasin （YL11）以及活化態prostasin （YL89）的表現差異 56
陸、皮膚幹細胞標記蛋白K15的表現差異 56
第五節 組織內活化態matriptase、HAI-1以及高醣基化HAI-2的相對位置 56
壹、活化態matriptase的不同表現位置 57
貳、活化態matriptase、HAI-1以及高醣基化HAI-2在相同組織上的表現 57
參、一個在基底層的細胞膜上表現的例子 58
第六節 比較糖尿病慢性傷口癒合良好或不佳的大組織中蛋白質的表現差異 59
壹、total matriptase （M24）的表現差異 59
貳、活化態matriptase （M69）的表現差異 60
參、HAI-1 （M19）的表現差異 60
肆、高醣基化HAI-2 （DC16）的表現差異 61
伍、total prostasin （YL11）以及活化態prostasin （YL89）的表現差異 61
陸、皮膚幹細胞標記蛋白K15的表現差異 61
柒、在棘狀層上方或顆粒層的活化態matriptase、HAI-1以及高醣基化HAI-2 62
第七節 慢性傷口癒合良好或不佳中缺氧現象與血管分布的關係 62
壹、缺氧現象和血管分布的關係 63
貳、糖尿病慢性傷口癒合良好與不佳的缺氧現象與血管分布 64
參、多次清創的糖尿病慢性傷口癒合良好與不佳的缺氧現象與血管分布 64
肆、糖尿病慢性傷口癒合良好與不佳的大組織中缺氧現象與血管分布 65
第八節 prostasin與角質細胞分化的關係 65
壹、prostasin在慢性傷口的表現位置與正常皮膚的差異 65
貳、角質細胞進行終端分化時的細胞型態差異 66
參、prostasin和matriptase等蛋白質在角質細胞終端分化時的表現差異 66
第九節 matriptase與高醣基化HAI-2的關係 67
壹、慢性傷口中活化態的matriptase與高醣基化HAI-2的位置 67
貳、正常角質細胞中活化態的matriptase與高醣基化HAI-2的關係 67
第四章 討論 69
第一節 臨床結果中慢性傷口癒合良好和不佳的定義 69
第二節 臨床結果都是傷口癒合良好但糖尿病足與糖尿病潰瘍的差異 69
第三節 洗腎對糖尿病潰瘍癒合良好或不佳中蛋白質的表現差異 70
第四節 在糖尿病潰瘍傷口中缺氧現象與血管新生的關係 70
第五節 終端分化與prostasin的關係 71
第六節 活化態matriptase與高醣基化HAI-2的關係 72
第七節 未來研究方向 72
第五章 結論 74
第六章 文獻參考 75

表目錄
表 1、一次清創的糖尿病足各蛋白質的判讀 84
表 2、一次清創的糖尿病潰瘍各蛋白質的判讀 84
表 3、多次清創下的糖尿病足各蛋白質的判讀 85
表 4、多次清創下的糖尿病潰瘍各蛋白質的判讀 86
表 5、一次清創下的伴隨洗腎的糖尿病足和手指各蛋白質的判讀 86
表 6、糖尿病慢性傷口的組織中活化態 matriptase （M69）、HAI-1 （M19）和高醣基化HAI-2 （DC16）在相似位置上的判讀 87
表 7、糖尿病慢性傷口的大組織中活化態 matriptase （M69）、HAI-1 （M19）和高醣基化HAI-2 （DC16）在相似位置上的判讀 88
表 8、糖尿病慢性傷口癒合良好或不佳的大組織中的蛋白質的表現判讀 89
表 9、糖尿病慢性傷口癒合良好與不佳多次清瘡前後的血管內皮細胞標記蛋白（EDG）的面積和比例 90
表 10、糖尿病慢性傷口癒合良好與不佳的大組織中血管內皮細胞標記蛋白（EDG）的表現面積和比例 91

圖目錄
圖 1、人類皮膚解剖圖 92
圖 2、人類表皮層的分層結構圖 93
圖 3、表皮層細胞各層之分化標誌蛋白（differentiation markers） 94
圖 4、matriptase、prostasin 與 HAI-1 的結構 95
圖 5、HAI-1 & HAI-2的結構比較 96
圖 6、matriptase的活化機轉與HAI-1之間的關係 97
圖 7、比較治療前後活化態matriptase的表現量 98
圖 8、免疫組織化學染色判讀-強度 99
圖 9、免疫組織化學染色判讀-範圍 100
圖 10、免疫組織化學染色判讀-表現比例 101
圖 11、Image J計算染色面積 102
圖 12、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中total matriptase （M24）的比較 103
圖 13、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中活化態 matriptase （M69）的比較 104
圖 14、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中HAI-1 （M19）的比較 105
圖 15、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中高醣基化的HAI-2 （DC16）的比較 106
圖 16、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中 total prostasin （YL11）的比較 107
圖 17、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中活化態 prostasin （YL89）的比較 108
圖 18、糖尿病足以及糖尿病潰瘍一次清創後傷口癒合良好或不佳中皮膚幹細胞標記蛋白K15的比較 109
圖 19、糖尿病足多次清創前後傷口癒合良好或不佳中total matriptase （M24）的比較 110
圖 20、糖尿病足多次清創前後傷口癒合良好或不佳中活化態 matriptase （M69）的比較 111
圖 21、糖尿病足多次清創前後傷口癒合良好或不佳中HAI-1 （M19）的比較 112
圖 22、糖尿病足多次清創前後傷口癒合良好或不佳中高醣基化HAI-2 （DC16）的比較 113
圖 23、糖尿病足多次清創前後傷口癒合良好或不佳中total prostasin （YL11）的比較 114
圖 24、糖尿病足多次清創前後傷口癒合良好或不佳中活化態 prostasin （YL89）的比較 115
圖 25、糖尿病足多次清創前後傷口癒合良好或不佳中皮膚幹細胞標記蛋白K15的比較 116
圖 26、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的total matriptase （M24）表現差異 117
圖 27、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的活化態matriptase （M69）表現差異 118
圖 28、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的HAI-1（M19）表現差異 119
圖 29、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的高醣基化HAI-2 （DC16）表現差異 120
圖 30、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的total prostasin （YL11）表現差異 121
圖 31、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的活化態 prostasin （YL89）表現差異 122
圖 32、比較在糖尿病足與糖尿病潰瘍多次清創前後傷口癒合良好的皮膚幹細胞標記蛋白K15表現差異 123
圖 33、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的total matriptase （M24）表現差異 124
圖 34、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的活化態 matriptase （M69）表現差異 125
圖 35、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的HAI-1 （M19）表現差異 126
圖 36、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的高醣基化HAI-2 （DC16）表現差異 127
圖 37、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的total prostasin （YL11）表現差異 128
圖 38、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的活化態 prostasin （YL89）表現差異 129
圖 39、比較在有無洗腎狀況下糖尿病足和手指潰瘍一次清創中傷口癒合良好或不佳的皮膚幹細胞標記蛋白K15表現差異 130
圖 40、組織內活化態matriptase （M69）表現在不同位置上 131
圖 41、組織內活化態matriptase、HAI-1以及高醣基化HAI-2會表現在上方棘狀層或者顆粒層的細胞膜上 132
圖 42、一個組織內活化態matriptase、HAI-1以及高醣基化HAI-2會同時表現在基底層的細胞膜上 133
圖 43、比較糖尿病慢性傷口癒合良好或不佳的大組織中HE stain圖的組織型態 134
圖 44、比較糖尿病慢性傷口癒合良好或不佳的大組織中total matriptase （M24）的表現 135
圖 45、比較糖尿病慢性傷口癒合良好或不佳的大組織中活化態 matriptase （M69）的表現 136
圖 46、比較糖尿病慢性傷口癒合良好或不佳的大組織中HAI-1 （M19）的表現 137
圖 47、比較糖尿病慢性傷口癒合良好或不佳的大組織中高醣基化HAI-2 （DC16）的表現 138
圖 48、比較糖尿病慢性傷口癒合良好或不佳的大組織中total prostasin （YL11）的表現 139
圖 49、比較糖尿病慢性傷口癒合良好或不佳的大組織中活化態 prostasin （YL89）的表現 140
圖 50、比較糖尿病慢性傷口癒合良好或不佳的大組織中皮膚幹細胞標記蛋白K15的表現 141
圖 51、比較糖尿病慢性傷口癒合良好和不佳的大組織中的蛋白質表現統計圖 142
圖 52、比較糖尿病慢性傷口癒合良好的大組織中活化態matriptase （M69）、HAI-1 （M19）以及高醣基化HAI-2 （DC16）的表現 143
圖 53、比較糖尿病慢性傷口癒合不佳的大組織中活化態matriptase （M69）、HAI-1 （M19）以及高醣基化HAI-2 （DC16）的表現 144
圖 54、細胞的氧氣充足或缺氧現象時HIF-1α的表現變化 145
圖 55、糖尿病慢性傷口中異常HIF-1α和EDG的染色對比 146
圖 56、糖尿病慢性傷口癒合良好或不佳的HIF-1α和EDG的染色對比 147
圖 57、糖尿病慢性傷口癒合良好中多次清創前後的HIF-1α和EDG的染色對比 148
圖 58、糖尿病慢性傷口癒合不佳中多次清創前後的HIF-1α和EDG的染色對比 149
圖 59、糖尿病慢性傷口癒合良好的大組織中HIF-1α和EDG的染色對比 150
圖 60、糖尿病慢性傷口癒合不佳的大組織中HIF-1α和EDG的染色對比 151
圖 61、糖尿病慢性傷口EDG的染色統計圖 152
圖 62、total prostasin （YL11）在正常皮膚與慢性傷口的表現差異 153
圖 63、在不同處理下HaCaT細胞的組織型態變化 154
圖 64、換成高鈣濃度培養基前後細胞中蛋白質的變化 155
圖 65、細胞密度逐漸增加時細胞中蛋白質的變化 156
圖 66、測試免疫沉澱法beads的結果 157
圖 67、在不同處理下細胞中蛋白質經過免疫沉澱法前後的matriptase （M24、M69）的變化 158
圖 68、在不同處理下細胞中蛋白質經過免疫沉澱法前後的HAI-1 （M19）的變化 159
圖 69、在不同處理下細胞中蛋白質經過免疫沉澱法前後的高醣基化HAI-2 （DC16）的變化 160

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