臺灣博碩士論文加值系統

Matriptase為首次於1993年在乳癌細胞株 (T47D)中發現具有分解胞外基質 (extracellular matrix)能力的蛋白酶，並於1999年首次自人類乳汁中將matriptase純化出來。matriptase經由S1P (sphingosine-1-phosphate) 的作用，變成具活性的蛋白酶；另外HAI-1 會與matriptase形成可逆性結合以調控其活性。研究顯示，matriptase具有活化HGF及uPA的能力，而這些蛋白質與癌細胞的轉移與浸潤有關。另外在一些上皮細胞癌 (carcinoma) 的研究，包括乳癌、攝護腺癌、卵巢癌、大腸癌、子宮頸癌等，觀察到matriptase的過度表現，或者matriptase與HAI-1的表現量比異常。經由這些發現我們要進一步來探討matriptase及HAI-1在位居國人惡性腫瘤十大死因之ㄧ的胃癌中扮演的角色。我們利用西方點墨法分析matriptase與HAI-1在人類胃腺癌細胞株（AGS）的表現以及對於胎牛血清與S1P的反應性。結果在實驗中發現，S1P會刺激人類胃腺癌細胞株（AGS）增加matriptase與HAI-1的表現；而在活化matriptase方面似乎沒有預期效果。相對的利用增加血清濃度的方式促進matriptase的活化卻有明顯的效果，顯示出在血清中，除了S1P以外，似乎有另外一個因子與AGS胃腺癌細胞株的matriptase活化有關。我們另外與三總一般外科陳醫師合作，利用免疫組織染色分析胃的腫瘤及非腫瘤樣本，從這些樣本的觀察與分析，我們發現在胃壁各層的癌細胞都持續表現matriptase。跟非腫瘤區域組織切片比較，活化態的matriptase主要表現在腫瘤組織；與非腫瘤區域相比，matriptase總量並未明顯的增加，不過可以觀察到在兩個區域的分布有明顯的差異。另外我們觀察到HAI-1在腫瘤的表現與非腫瘤區域相比有相當程度的增加。

Matriptase is a novel matrix-degrading serine protease identified from T-47D human breast cancer cells. Matriptase was purified from human breast milk as a complx with a Kunitz-type serine protease inhibitor, termed hepatocyte growth factor activator inhibitor-1(HAI-1). HAI-1 is not only a matriptase-binding protein, but also a potential inhibitor of matriptase. Matriptase could function as a membrane catalyst on cell surfaces to recruit and activate urokinase-type plasminogen activator, an important extracellular matrix-degrading protease, and hepatocyte growth factor, an important cell motility factor. The activation of matriptase is regulated by sphingosine-1-phosphate. According to the literature, we have learned that matriptase may play a crucial role in cancer metastasis. In breast cancer, high-level expression of matriptase were associated with poor outcome. Since gastric cancer is one of the top 10 cancer leading causes of death in Taiwan, we chose to study the role of matriptase and HAI-1 in gastric cancer. In our study, gastric specimen are collected from the department of the general surgery of TSGH. We utilized immunohistochemistry staining to analyze the expression level and distribution of matriptase and HAI-1. We also utilized cultured cells to mimic different conditions in the literature. We further examined those cells by western analysis. From the results of western blotting, we observed S1P can induce the expression of matriptase and HAI-1 in cultured cells. Our results suggest that besides S1P, FBS could contain other factors to activate matriptase in AGS tumor cell line. In immunohistochemistry, we observed active form matriptase was primarily express in tumor area, and the distribution of matriptase and HAI-1 in tumor area is different from non-tumor area. We also found the level of HAI-1 in tumor area is higher than non-tumor area.

目 錄
頁 次
目 錄....................................................................................................................I
圖目錄..................................................................................................................V
縮寫表...............................................................................................................VII
中文摘要..........................................................................................................VIII
英文摘要..............................................................................................................X
第一章緒論...........................................................................................................1
第一節 蛋白酶（Proteases）.......................................................................1
第二節 蛋白酶（Proteases）、胞外基質（extracellular matrix）在癌症浸 潤（invasion）與轉移(metastasis)的過程中扮演的角色.............2
第三節 來自上皮細胞的蛋白酶（Epithelial-derived protease）..............3
第四節 Matriptase的表現、活化與組織分佈.............................................4
壹、血清濃度的改變造成matriptase 的活化及釋出（shedding）....4
貳、活化態的matriptase具有活化pro-HGF及pro-uPA的能力............5
参、Matriptase與HAI-1在人類正常乳房上皮細胞及各類乳癌細胞的表現與上皮細胞標記（epithelial marker）具有一致性...............6
肆、血清中的脂蛋白 (lipoprotein)與matriptase的活化有關..............7
I
第五節 HAI-1 的結構與功能......................................................................8
壹、HAI-1 的結構.................................................................................8
貳、HAI-1 的功能.................................................................................9
参、HAI-1 在調控matriptase 活化的過程中所扮演的角色..............9
第六節 根據matriptase及HAI-1間的關係所發展出的抗體....................11
第七節 matriptase 可能扮演的生理角色.................................................12
第八節 在各類癌症中觀察到matriptase或HAI-1的異常........................15
壹、matriptase/HAI-1與乳癌的關係..................................................15
貳、matriptase/HAI-1與卵巢癌的關係..............................................15
参、matriptase/HAI-1與子宮頸癌的關係..........................................16
肆、matriptase/HAI-1與攝護腺癌的關係..........................................17
伍、matriptase只有在上皮來源（epithelial origin）的腫瘤可以看到表現................................................................................................17
第九節 胃及胃癌的形成、分類、流行病學（epidemiology）..............18
壹、胃的解剖構造及胃壁組成...........................................................18
貳、胃癌的流行病學、致病因子及分類...........................................19
第十節 實驗目的........................................................................................23
第二章實驗材料與方法.....................................................................................24
實驗材料......................................................................................................24
II
壹、藥品試劑.......................................................................................24
貳、實驗儀器.......................................................................................26
参、緩衝液及SDS-PAGE 之備製......................................................26
肆、sample 之取得與保存..................................................................28
實驗方法......................................................................................................28
壹、冷凍切片.......................................................................................28
貳、免疫組織化學染色.......................................................................29
参、蘇木紫-伊紅染色 (Hematoxylin & Eosin stain).........................31
肆、人類胃腺癌細胞株(AGS)之細胞培養........................................31
伍、西方點墨法...................................................................................31
第三章結果.........................................................................................................34
第一節、S1P影響Matriptase在人類胃腺癌細胞株(AGS)的表現與活化34
第二節、在胃壁各層的癌細胞都可以觀察到matriptase的表現.............35
第三節、HAI-1的表現與否隨這癌細胞侵犯深度產生變化..................35
第四節、觀察腫瘤及鄰近非腫瘤區域的組織切片，發現在腫瘤區域的 matriptase、HAI-1的分佈產生差異.........................................37
第五節、活化態的matriptase主要表現在腫瘤區域.................................39
第六節、在腫瘤鄰近的非腫瘤區域所表現的matriptase主要為單鏈型.39
第四章討論.........................................................................................................41
III
第一節 Sphingosine 1-phosphate（S1P）似乎具有刺激人類胃腺癌細胞（AGS）株表現matriptase及HAI-1的作用.................................41
第二節 在胃癌形成過程中，matriptase與HAI-1的表現及分佈發生漸進 式的變化........................................................................................42
第五章參考文獻.................................................................................................46
IV
圖目錄
圖一、matriptase與HAI-1及相關抗體....................................................50
圖二、胃壁縱切圖......................................................................................51
圖三、血清濃度的增加造成matriptase與HAI-1 複合物釋放至培養液中.........................................................................................................52
圖四、在維持一定濃度的FBS情況下，不具活性的matriptase與Matriptase與HAI-1 複合物維持一定的比例.............................53
圖五、S1P誘導Matriptase的表現與活化...............................................54
圖六、侵犯到黏膜肌層的胃癌切片組織染色..........................................55
圖七、侵犯到黏膜下層的胃癌切片組織染色..........................................56
圖八、侵犯到肌肉層的胃癌切片組織染色..............................................57
圖九、侵犯到漿膜層的胃癌切片組織染色..............................................58
圖十、淋巴上皮癌的組織切片染色..........................................................59
圖十一、侵犯到黏膜肌層的胃癌，胃癌細胞僅表現matriptase，未表現 HAI-1...........................................................................................60
圖十二、侵犯到黏膜肌層的胃癌，同時表現matriptase及HAI-1........61
圖十三、侵犯到黏膜下層的胃癌，只有黏膜表面細胞有表現HAI-1..62
圖十四、侵犯到黏膜下層的胃癌，在黏膜下層的癌細胞不表現HAI-1.........................................................................................................63
圖十五、侵犯到肌肉層的胃癌，在肌肉層的癌細胞可以看到matriptase
V
及HAI-1的表現..........................................................................64
圖十六、在同一個腫瘤組織切片中，matriptase 及HAI-1在肌肉層及黏 膜層的表現差異。......................................................................67
圖十八、利用抗體M32比較matriptase在非腫瘤及腫瘤區域的分佈。.........................................................................................................72
圖十九、利用抗體M19可以在腫瘤的組織切片偵測到HAI-1的表現。.........................................................................................................73
圖二十、利用抗體M19在非腫瘤區域的組織切片幾乎偵測不到HAI-1 的表現。......................................................................................74
圖二十一、利用西方點墨法觀察非腫瘤及腫瘤區域HAI-1的表現。.75
圖二十二、利用西方點墨法觀察非腫瘤及腫瘤區域matriptase的表現76
圖二十四、在有表現活化態的matriptase的位置，抗體M19偵測不到 HAI-1的表現..........................................................................78
圖二十五、在腫瘤鄰近的非腫瘤區域所表現的matriptase主要為單鏈型.........................................................................................................80

1.Benaud, C., Dickson, R. B. & Lin, C. Y. Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor. Eur J Biochem 268, 1439-47 (2001).

2.Benaud, C. et al. Sphingosine 1-phosphate, present in serum-derived lipoproteins, activates matriptase. J Biol Chem 277, 10539-46 (2002).

3.Benaud, C. M., Oberst, M., Dickson, R. B. & Lin, C. Y. Deregulated activation of matriptase in breast cancer cells. Clin Exp Metastasis 19, 639-49 (2002).

4.Bhatt, A. S. et al. Quantitation of membrane type serine protease 1 (MT-SP1) in transformed and normal cells. Biol Chem 384, 257-66 (2003).

5.Crew, K. D. & Neugut, A. I. Epidemiology of gastric cancer. World J Gastroenterol 12, 354-62 (2006).

6.Denda, K., Shimomura, T., Kawaguchi, T., Miyazawa, K. & Kitamura, N. Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 1. J Biol Chem 277, 14053-9 (2002).

7.Everett, S. M. & Axon, A. T. Early gastric cancer: disease or pseudo-disease? Lancet 351, 1350-2 (1998).

8.Hoang, C. D. et al. Gene expression profiling identifies matriptase overexpression in malignant mesothelioma. Chest 125, 1843-52 (2004).

9.Johnson, M. D., Oberst, M. D., Lin, C. Y. & Dickson, R. B. Possible role of matriptase in the diagnosis of ovarian cancer. Expert Rev Mol Diagn 3, 331-8 (2003).

10.Kang, J. Y. et al. Tissue microarray analysis of hepatocyte growth factor/Met pathway components reveals a role for Met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer. Cancer Res 63, 1101-5 (2003).

11.Kataoka, H., Miyata, S., Uchinokura, S. & Itoh, H. Roles of hepatocyte growth factor (HGF) activator and HGF activator inhibitor in the pericellular activation of HGF/scatter factor. Cancer Metastasis Rev 22, 223-36 (2003).

12.Lee, J. W. et al. Increased expression of matriptase is associated with histopathologic grades of cervical neoplasia. Hum Pathol 36, 626-33 (2005).

13.Lee, M. S., Kiyomiya, K., Benaud, C., Dickson, R. B. & Lin, C. Y. Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells. Am J Physiol Cell Physiol 288, C932-41 (2005).

14.Lee, S. L., Dickson, R. B. & Lin, C. Y. Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease. J Biol Chem 275, 36720-5 (2000).

15.Lin, C. Y., Anders, J., Johnson, M. & Dickson, R. B. Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk. J Biol Chem 274, 18237-42 (1999).

16.Lin, C. Y., Anders, J., Johnson, M., Sang, Q. A. & Dickson, R. B. Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. J Biol Chem 274, 18231-6 (1999).

17.Lin, C. Y. et al. Characterization of a novel, membrane-bound, 80-kDa matrix-degrading protease from human breast cancer cells. Monoclonal antibody production, isolation, and localization. J Biol Chem 272, 9147-52 (1997).

18.List, K. et al. Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis. Oncogene 21, 3765-79 (2002).

19.List, K. et al. Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation. Genes Dev 19, 1934-50 (2005).

20.List, K. et al. Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1. J Cell Biol 163, 901-10 (2003).

21.Oberst, M. et al. Matriptase and HAI-1 are expressed by normal and malignant epithelial cells in vitro and in vivo. Am J Pathol 158, 1301-11 (2001).

22.Oberst, M. D. et al. HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease. Am J Physiol Cell Physiol 289, C462-70 (2005).

23.Oberst, M. D. et al. Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: correlation with clinical outcome and tumor clinicopathological parameters. Clin Cancer Res 8, 1101-7 (2002).

24.Oberst, M. D. et al. Characterization of matriptase expression in normal human tissues. J Histochem Cytochem 51, 1017-25 (2003).

25.Pyne, S. & Pyne, N. J. Sphingosine 1-phosphate signalling in mammalian cells. Biochem J 349, 385-402 (2000).

26.Saleem, M. et al. A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1. Cancer Epidemiol Biomarkers Prev 15, 217-27 (2006).

27.Santin, A. D. et al. The novel serine protease tumor-associated differentially expressed gene-15 (matriptase/MT-SP1) is highly overexpressed in cervical carcinoma. Cancer 98, 1898-904 (2003).

28.Shi, Y. E. et al. Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells. Cancer Res 53, 1409-15 (1993).

29.Tanimoto, H. et al. Ovarian tumor cells express a transmembrane serine protease: a potential candidate for early diagnosis and therapeutic intervention. Tumour Biol 22, 104-14 (2001).

30. 吳邱文等主編 胃癌之診斷及治療第一版2000年.

31. 日本胃癌協會編 Japanese Classification of Gastric Carcinoma, June 1999, 13th Edition