臺灣博碩士論文加值系統

matriptase是屬於第二型嵌膜絲胺酸蛋白酶 (type II transmembrane serine protease, TTSP) ，會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因子抑制蛋白 (hepatocyte growth factor activator inhibitor 1, HAI-1) ，也是matriptase的結合蛋白，在matriptase活化後會迅速地結合，藉以調控matriptase的活性。在動物實驗中發現，過度表現matriptase的基因轉殖鼠會造成上皮腫瘤，形成自發性的鱗狀上皮細胞癌；反之將小鼠的matriptase基因剔除，則會導致小鼠的皮膚失去保水的功能，同時毛髮發育不良無法突破表皮生長，皮膚也會有觀察到紅腫、皺縮、乾燥的現象。由此可以推測，matriptase和皮膚分化與上皮細胞的增生中扮演著重要的角色。除此之外matriptase也參與了許多跟增生分化有關的重要訊息傳遞路徑，這在皮膚傷口癒合中也佔了一個很重要的過程，例如matriptase在之前文獻中得知可以切割pro-HGF使其活化進而結合下游受器c-Met，在傷口癒合過程中，c-Met也扮演著重要的角色。因此在本研究中，藉由取得相對正常皮膚組織跟糖尿病患者足部潰瘍後未接受治療及治療後的周邊皮膚組織，來探討matriptase在兩者中的表現差異，並觀察其他相關蛋白的表現有何異同。而在此次的實驗結果中可以看到，在同樣是足部皮膚中在型態上，可以發現：(1) 糖尿病足潰瘍周邊組織較正常皮膚組織的表皮層型態較為不同，且皮膚中的附屬器也比正常皮膚少，汗腺也有觀察到較為萎縮的現象。(2) 正常皮膚與糖尿病足潰瘍周邊皮膚中，matriptase表現主要分布是由表皮層的基底層向上遞減，而正常皮膚中HAI-1的表現則是分布於整個表皮層，但在糖尿病足潰瘍周邊皮膚中的檢體中發現，大部分的組別會有往上遞減或者較弱的表現。(3) 與此次的實驗中，正常皮膚約有40%比例有觀察到活化態matriptase表現於基底層位置，而糖尿病足潰瘍檢體在表皮層活化的matriptase有高達76%左右的機率被觀察到在基底層位置，而c-Met (HGF receptor)則在兩種皮膚中都看到有表現在基底層。(4) 與正常皮膚不同的是，K15在之前研究中常表現在正常皮膚表皮層的基底層，但在慢性傷口周邊皮膚表皮層則完全看不到K15的表現。(6)在接受過治療的檢體中，在蛋白質的表現上差異並不大。(7)非糖尿病的慢性傷口，matriptase與相關蛋白的表現類似於糖尿病足潰瘍相比，有高度的相似性。根據以上實驗結果，也許在慢性傷口之中，HAI-1的表現下降可能會影響傷口癒合的能力以及許多本來抑制的蛋白酶失去控制，而matriptase的活化機率較高被觀察到，也許也和HAI-1的下降而無法有效地抑制有關，可是是否真的為造成傷口無法癒合的原因，其中詳細的機制仍有待後續研究。

Matriptase, a member of the type II transmembrane serine proteases, is widely expressed in all epithelia. After activation, matriptase is bound rapidly by its cognate inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Previous study has shown matriptase knock-out mice exhibited abnormal development of epidermis and hair follicle. matriptase overexpression of matriptase in the skin of transgenic mice may trigger spontaneous squamous cell carcinoma. Therefore it suggests that matriptase palys an important role in epidermis differentiation and hair follicle development Recent studies have shown that matriptase participates in epithelia growth and morphogenesis through HGF activation, HGF receptor (c-Met) binding , and trigger this signaling, which is critical in wound healing. In this study, we use immunohistochemistry to investigate the expression of matriptase in normal and diabetic ulcer foot skin. We found different morphology and expression pattern of HAI-1, K15, and c-Met between normal and diabetic patients’s foot skin. We conclude that: (1) In morphology, diabetic patients’ epidermis is different from normal, and the sweat gland look atrophy. (2) In normal skin and diabetic ulcer, matriptase is expressed decreasing along with the progression of differentiation from the stratum basal to the stratum granulosum. HAI-1 is expressed evenly in normal epidermis, but HAI-1 is expressed much weaker in most diabetic ulcer skin. (3) In this study, 6 of 15 normal samples were positive for activated matriptase (40%), but 32 of 42 diabetic samples were positive (76%). Normal and diabetic skin samples show similiar expression pattern of c-Met in basal layer. (4) K15 expression in normal skin is located at epidermis’s basal layer, but in diabetic foot skin, we could not detect K15 expression. (5) Diabetic foot ulcer skin samples show similiar result in both before treatment and after treatment groups. (6) Diabetic free chronic wound, show similar expression pattern like diabetic foot ulcer. Our results suggest that, low level of HAI-1 could be related to slow wound healing efficiency and its inhibition activity for other protease is out of control. The activation of matriptase in diabetic foot ulcer could be induced by the decrease of HAI-1. However the detailed mechanism needs to be further confirmed.

表目錄 I
圖目錄 II
縮寫表 IV
摘要 V
Abstract VII
第一章 緒論 1
第一節 Matriptase 1
一、Matriptase的發現 1
二、Matriptase的結構 1
三、Matriptase的分布與生理功能 2
第二節、HAI-1 5
一、 HAI-1的發現 5
二、 HAI-1的結構 5
三、 HAI-1的分布與功能 6
第三節 Matriptase與HAI-1 8
一、 matriptase的活化 8
二、 matriptase可能的活化機制 9
三、 matriptase的下游反應 10
四、 Matriptase與HAI-1之間的平衡 12
第四節、皮膚 13
一、皮膚的基本構造 13
二、表皮層的結構分層 15
三、皮膚的附屬器官（skin appendages） 17
四、上皮幹細胞（keratinocyte stem cell） 19
第五節、傷口癒合 (wound healing) 20
一、 傷口癒合 (wound healing) 20
二、慢性傷口 (chronic wound) 22
三、 糖尿病足潰瘍 (diabetic foot ulcer) 22
第六節 實驗目的 24
第二章　材料與方法 25
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
第三節　實驗方法 28
一、檢體處理、冷凍包埋 28
二、冷凍切片(Frozen section) 28
三、蘇木紫與伊紅染色 (Hematoxylin and eosin stain) 29
四、免疫組織化學染色 (Immunohistochemistry analysis) 29
第三章 結果 31
第一節 Matriptase和HAI-1在正常皮膚與糖尿病足潰瘍傷口的表現 31
一、 表皮 31
二、 汗腺 32
第二節 活化態matriptase、c-Met及K15在正常皮膚以及糖尿病足潰瘍皮膚的表現分布 33
一、 活化態matriptase在正常皮膚與糖尿病足潰瘍周邊傷口的分布： 33
二、 c-Met在正常皮膚與糖尿病足潰瘍周邊傷口的表現分布： 33
三、 K15在正常皮膚與糖尿病足潰瘍的表現分布 34
第三節 比較未治療前以及有接受治療後，糖尿病足潰瘍中matriptase及相關蛋白的分布與表現 35
一、在治療前後的糖尿病足潰瘍皮膚，matriptase與HAI-1的分布 35
二、在治療前後的糖尿病足潰瘍皮膚，活化態 matriptase與c-Met與K15的分布 36
三、同一病人，依傷口嚴重程度不同的檢體來比較matriptase及相關蛋白的表現情形 37
第四節、非糖尿病慢性傷口中，觀察matriptase及相關蛋白的表現 38
第四章 討論 41
第一節 正常皮膚組織與糖尿病足潰瘍組織的表現差異 41
第二節、比較治療前後的糖尿病足潰瘍傷口的表現差異 43
第三節、非糖尿病的慢性傷口，matritpase的表現相類似 44
第四節 結論 47
第五節 未來實驗方向 48
第五章 文獻參考 49


表目錄
表 一、正常皮膚與糖尿病足潰瘍皮膚組織的免疫組織染色統計 54

圖目錄
圖 一、Matriptase與HAI-1之結構圖 54
圖 二、Matriptase與HAI-1從成熟、活化、形成複合物到最後從膜上脫落的流程圖 55
圖 三、皮膚的結構圖 56
圖 四、表皮層的結構圖 57
圖 五、正常皮膚下，HAI-1與matriptase在表皮層上的分布 (400X) 58
圖 六、正常皮膚中，HAI-1與matriptase在汗腺中的表現(400X) 59
圖 七、在糖尿病足潰瘍中，HAI-1與matriptase在表皮層上的表現分布情形 (100X)， 60
圖 八、在糖尿病足潰瘍中，HAI-1與matriptase在表皮層上的表現分布情形 (400X)： 61
圖 九、在糖尿病足潰瘍中，HAI-1與matriptase在汗腺中的表現(400X) 62
圖 十、正常皮膚中，活化態matriptase的表現(400X) 63
圖 十一、在糖尿病足潰瘍中，活化態matriptase的表現(100X) 64
圖 十二、糖尿病足潰瘍中，活化態matriptase的表現(400X) 65
圖 十三、正常皮膚中，c-Met在表皮層的表現(400X) 66
圖 十四、正常皮膚中，c-Met在汗腺的表現(400X) 67
圖 十五、糖尿病足潰瘍中，c-Met在表皮層的表現(400X) 68
圖 十六、糖尿病足潰瘍中，c-Met在汗腺的表現(400X) 69
圖 十七、正常皮膚中，K15在表皮層的表現(400X) 70
圖 十八、糖尿病足潰瘍中，K15在表皮層的表現(400X) 71
圖 十九、在治療前與治療後，negative control mouse IgG在糖尿病足潰瘍周邊傷口的表皮層表現(100X) 72
圖 二十、在未治療與有治療，negative control mouse IgG在糖尿病足潰瘍周邊傷口的表皮層表現(400X) 73
圖 二十一、在治療前與有治療後，HAI-1在糖尿病足潰瘍周邊傷口的表皮層表現(100X) 76
圖 二十二、在未治療前與有治療後，HAI-1在糖尿病足潰瘍周邊傷口的表皮層表現(400X) 76
圖 二十三、在治療前與有治療後，matriptase在糖尿病足潰瘍周邊傷口的表皮層表現(100X) 79
圖 二十四、在治療前與治療後，matriptase在糖尿病足潰瘍周邊傷口的表皮層表現(400X) 79
圖 二十五、在治療前與有治療後，活化態matriptase在糖尿病足潰瘍周邊傷口的表皮層表現(100X) 80
圖 二十六、在治療前與有治療後，活化態matriptase在糖尿病足潰瘍周邊傷口的表皮層表現(400X) 81
圖 二十七、在治療前與有治療後，c-Met在糖尿病足潰瘍周邊傷口的表皮層表現(100X) 82
圖 二十八、在治療前與有治療後，c-Met在糖尿病足潰瘍周邊傷口的表皮層表現(400X) 83
圖 二十九、在治療前與有治療後，K15在糖尿病足潰瘍周邊傷口的表皮層表現(100X) 84
圖 三十、在治療前與有治療後，K15在糖尿病足潰瘍周邊傷口的表皮層表現(400X) 85
圖 三十一、以HE染色觀察比較同一個病人，左右足不同嚴重程度的傷口周邊皮膚的組織型態(40X) 86
圖 三十二、比較同一個病人，左右足不同嚴重程度的傷口周邊皮膚matriptase與HAI-1的表現(100X) 87
圖 三十三、比較同一個病人，左右足不同嚴重程度的傷口周邊皮膚matriptase與HAI-1的表現(400X) 88
圖 三十四、比較同一個病人，左右足不同嚴重程度的傷口周邊皮膚活化態matriptase、c-Met與K15的表現(100X) 89
圖 三十五、比較同一個病人，左右足不同嚴重程度的傷口周邊皮膚活化態matriptase、c-Met與K15的表現(400X) 90
圖 三十六、非糖尿病慢性傷口，HAI-1與matriptase的表現情形(100X) 91
圖 三十七、非糖尿病慢性傷口，HAI-1與matriptase的表現情形(400X) 92
圖 三十八、非糖尿病慢性傷口，活化態matriptase、c-Met與K15的表現情形(100X) 93
圖 三十九、非糖尿病慢性傷口，活化態matriptase、c-Met與K15的表現情形(400X) 94

1.Shi YE, Torri J, Yieh L, Wellstein A, Lippman ME, Dickson RB: Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells. Cancer research 1993, 53(6):1409-1415.
2.Lin CY, Wang JK, Torri J, Dou L, Sang QA, Dickson RB: Characterization of a novel, membrane-bound, 80-kDa matrix-degrading protease from human breast cancer cells. Monoclonal antibody production, isolation, and localization. The Journal of biological chemistry 1997, 272(14):9147-9152.
3.Lin CY, Anders J, Johnson M, Sang QA, Dickson RB: Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. The Journal of biological chemistry 1999, 274(26):18231-18236.
4.Lin CY, Anders J, Johnson M, Dickson RB: Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk. The Journal of biological chemistry 1999, 274(26):18237-18242.
5.Kim MG, Chen C, Lyu MS, Cho EG, Park D, Kozak C, Schwartz RH: Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains. Immunogenetics 1999, 49(5):420-428.
6.Takeuchi T, Shuman MA, Craik CS: Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue. Proceedings of the National Academy of Sciences of the United States of America 1999, 96(20):11054-11061.
7.Lin CY, Tseng IC, Chou FP, Su SF, Chen YW, Johnson MD, Dickson RB: Zymogen activation, inhibition, and ectodomain shedding of matriptase. Frontiers in bioscience : a journal and virtual library 2008, 13:621-635.
8.Oberst MD, Singh B, Ozdemirli M, Dickson RB, Johnson MD, Lin CY: Characterization of matriptase expression in normal human tissues. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2003, 51(8):1017-1025.
9.List K, Haudenschild CC, Szabo R, Chen W, Wahl SM, Swaim W, Engelholm LH, Behrendt N, Bugge TH: Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis. Oncogene 2002, 21(23):3765-3779.
10.List K, Bugge TH, Szabo R: Matriptase: potent proteolysis on the cell surface. Molecular medicine (Cambridge, Mass) 2006, 12(1-3):1-7.
11.Chen YW, Wang JK, Chou FP, Wu BY, Hsiao HC, Chiu H, Xu Z, Baksh AN, Shi G, Kaul M et al: Matriptase regulates proliferation and early, but not terminal, differentiation of human keratinocytes. The Journal of investigative dermatology 2014, 134(2):405-414.
12.Shimomura T, Denda K, Kitamura A, Kawaguchi T, Kito M, Kondo J, Kagaya S, Qin L, Takata H, Miyazawa K et al: Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor. The Journal of biological chemistry 1997, 272(10):6370-6376.
13.Kirchhofer D, Peek M, Li W, Stamos J, Eigenbrot C, Kadkhodayan S, Elliott JM, Corpuz RT, Lazarus RA, Moran P: Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1. The Journal of biological chemistry 2003, 278(38):36341-36349.
14.Eigenbrot C, Ganesan R, Kirchhofer D: Hepatocyte growth factor activator (HGFA): molecular structure and interactions with HGFA inhibitor-1 (HAI-1). The FEBS journal 2010, 277(10):2215-2222.
15.Fan B, Wu TD, Li W, Kirchhofer D: Identification of hepatocyte growth factor activator inhibitor-1B as a potential physiological inhibitor of prostasin. The Journal of biological chemistry 2005, 280(41):34513-34520.
16.Kataoka H, Suganuma T, Shimomura T, Itoh H, Kitamura N, Nabeshima K, Koono M: Distribution of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in Human Tissues: Cellular Surface Localization of HAI-1 in Simple Columnar Epithelium and Its Modulated Expression in Injured and Regenerative Tissues. Journal of Histochemistry & Cytochemistry 1999, 47(5):673-682.
17.Kataoka H, Shimomura T, Kawaguchi T, Hamasuna R, Itoh H, Kitamura N, Miyazawa K, Koono M: Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Specific Cell Surface Binding Protein of Hepatocyte Growth Factor Activator (HGFA) and Regulates HGFA Activity in the Pericellular Microenvironment. Journal of Biological Chemistry 2000, 275(51):40453-40462.
18.Szabo R, Molinolo A, List K, Bugge TH: Matriptase inhibition by hepatocyte growth factor activator inhibitor-1 is essential for placental development. Oncogene 2007, 26(11):1546-1556.
19.Ovaere P, Lippens S, Vandenabeele P, Declercq W: The emerging roles of serine protease cascades in the epidermis. Trends in biochemical sciences 2009, 34(9):453-463.
20.Benaud C, Dickson RB, Lin CY: Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor. European journal of biochemistry / FEBS 2001, 268(5):1439-1447.
21.Kojima K, Inouye K: Activation of matriptase zymogen. Journal of biochemistry 2011, 150(2):123-125.
22.Benaud C, Oberst M, Hobson JP, Spiegel S, Dickson RB, Lin CY: Sphingosine 1-phosphate, present in serum-derived lipoproteins, activates matriptase. The Journal of biological chemistry 2002, 277(12):10539-10546.
23.Tseng IC, Xu H, Chou FP, Li G, Vazzano AP, Kao JP, Johnson MD, Lin CY: Matriptase activation, an early cellular response to acidosis. The Journal of biological chemistry 2010, 285(5):3261-3270.
24.Wang JK, Teng IJ, Lo TJ, Moore S, Yeo YH, Teng YC, Kaul M, Chen CC, Zuo AH, Chou FP et al: Matriptase autoactivation is tightly regulated by the cellular chemical environments. PloS one 2014, 9(4):e93899.
25.Lee SL, Dickson RB, Lin CY: Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease. The Journal of biological chemistry 2000, 275(47):36720-36725.
26.Jiang WG, Hiscox S: Hepatocyte growth factor/scatter factor, a cytokine playing multiple and converse roles. Histology and histopathology 1997, 12(2):537-555.
27.Kawaida K, Matsumoto K, Shimazu H, Nakamura T: Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proceedings of the National Academy of Sciences 1994, 91(10):4357-4361.
28.Conway K, Price P, Harding KG, Jiang WG: The molecular and clinical impact of hepatocyte growth factor, its receptor, activators, and inhibitors in wound healing. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society 2006, 14(1):2-10.
29.Chmielowiec J, Borowiak M, Morkel M, Stradal T, Munz B, Werner S, Wehland J, Birchmeier C, Birchmeier W: c-Met is essential for wound healing in the skin. The Journal of cell biology 2007, 177(1):151-162.
30.Takeuchi T, Harris JL, Huang W, Yan KW, Coughlin SR, Craik CS: Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. The Journal of biological chemistry 2000, 275(34):26333-26342.
31.Lee M-S: Matrix-Degrading Type II Transmembrane Serine Protease Matriptase: Its Role in Cancer Development and Malignancy; 2008.
32.Oberst MD, Chen LY, Kiyomiya K, Williams CA, Lee MS, Johnson MD, Dickson RB, Lin CY: HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease. American journal of physiology Cell physiology 2005, 289(2):C462-470.
33.Chou FP, Chen YW, Zhao XF, Xu-Monette ZY, Young KH, Gartenhaus RB, Wang JK, Kataoka H, Zuo AH, Barndt RJ et al: Imbalanced matriptase pericellular proteolysis contributes to the pathogenesis of malignant B-cell lymphomas. The American journal of pathology 2013, 183(4):1306-1317.
34.Webb A, Li A, Kaur P: Location and phenotype of human adult keratinocyte stem cells of the skin. Differentiation 2004, 72(8):387-395.
35.Pincelli C, Marconi A: Keratinocyte stem cells: friends and foes. Journal of cellular physiology 2010, 225(2):310-315.
36.Fuchs E, Horsley V: More than one way to skin. Genes & development 2008, 22(8):976-985.
37.Gantwerker EA, Hom DB: Skin: histology and physiology of wound healing. Clinics in plastic surgery 2012, 39(1):85-97.
38.Gurtner GC, Werner S, Barrandon Y, Longaker MT: Wound repair and regeneration. Nature 2008, 453(7193):314-321.
39.Akita S, Yoshimoto H, Akino K, Ohtsuru A, Hayashida K, Hirano A, Suzuki K, Yamashita S: Early experiences with stem cells in treating chronic wounds. Clinics in plastic surgery 2012, 39(3):281-292.
40.Boulton AJM, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J: The global burden of diabetic foot disease. The Lancet 2005, 366(9498):1719-1724.
41.Boulton AJ: The pathogenesis of diabetic foot problems: an overview. Diabet Med 1996, 13(1):S12-16.
42.Sen CK: Wound healing essentials: let there be oxygen. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society 2009, 17(1):1-18.
43.Itoh H, Kataoka H, Tomita M, Hamasuna R, Nawa Y, Kitamura N, Koono M: Upregulation of HGF activator inhibitor type 1 but not type 2 along with regeneration of intestinal mucosa. American journal of physiology Gastrointestinal and liver physiology 2000, 278(4):G635-643.
44.Miyazawa K, Shimomura T, Naka D, Kitamura N: Proteolytic activation of hepatocyte growth factor in response to tissue injury. The Journal of biological chemistry 1994, 269(12):8966-8970.
45.Waseem A, Dogan B, Tidman N, Alam Y, Purkis P, Jackson S, Lalli A, Machesney M, Leigh IM: Keratin 15 expression in stratified epithelia: downregulation in activated keratinocytes. The Journal of investigative dermatology 1999, 112(3):362-369.
46.Smiley AK, Klingenberg JM, Boyce ST, Supp DM: Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting. Burns 2006, 32(2):135-138.
47.Usui ML, Mansbridge JN, Carter WG, Fujita M, Olerud JE: Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2008, 56(7):687-696.
48.Chen YW, Wang JK, Chou FP, Chen CY, Rorke EA, Chen LM, Chai KX, Eckert RL, Johnson MD, Lin CY: Regulation of the matriptase-prostasin cell surface proteolytic cascade by hepatocyte growth factor activator inhibitor-1 during epidermal differentiation. The Journal of biological chemistry 2010, 285(41):31755-31762.