臺灣博碩士論文加值系統

中 文 摘 要
Hepsin為絲月安酸蛋白酵素的一種，常表現於多種細胞表面。它是一種第二型膜蛋白，在肝臟細胞的表現量最多。Hepsin與細胞的生長及轉移、肝細胞的發育、受精卵的著床以及血液凝固機轉有關。Hepsin基因剔除的小鼠並無異常，且生長及交配、繁殖也正常；至此，對hepsin的研究尚無法描繪出其特異的重要生理功能及機轉。本論文著眼於hepsin是否與凝血機轉有關，因此利用基因轉殖技術，在小鼠體內血管內皮細胞大量表達hepsin，俾使hepsin直接與血流中的凝固因子或其它蛋白質接觸，以觀察第七因子能否被高度活化而造成小鼠血栓。轉殖基因的表現是選用具有血管內皮細胞特異性的啟動子，Tie2 promoter，以調控hepsin的表現，除此之外，為了能在胚胎發育的各個時期分析表達hepsin的組織及細胞，因而在表達載體內同時嵌入含有IRES (internal ribosomal entry site)-NLS (nuclear localization signal)-LacZ的指標基因(reporter gene)。建構完成的載體首先須利用細胞株測試其正確性，本論文利用牛動脈內皮細胞株“BEC”，將構築的表達載體轉染至BEC後，利用X-gal染色，確可得到陽性細胞株。在轉殖小鼠實驗上，共注射FVB及C57BL/6兩種小鼠品系受精卵，初步的結果顯示，由南方點墨法證實轉殖基因確已嵌入小鼠且片段完整，而帶有轉殖基因的小鼠大多數在出生後24小時內死亡（在34隻陽性FVB小鼠中有18隻；在3隻陽性C57BL/6小鼠中全數），且在C57BL/6小鼠中27隻陰性小鼠全數存活，反觀注射hepsin活化區突變的載體於C57BL/6時，11隻帶有轉殖基因的小鼠只有1隻死亡， 9隻不帶有轉殖基因的小鼠也只有1隻死亡。這樣的結果推測與hepsin表現有關。
在分析小鼠胚胎發育上，存活的小鼠活體染色得知LacZ大量表現於E12.5的小鼠血管組織。在死亡的轉殖鼠病理組織上，並未有血栓的發生，因此須更詳細的分析來解釋小鼠死亡的原因。

ABSTRACT
Hepsin, an integral membrane serine proteinase, is produced in most of the tissues and particularly at high levels in the liver. The functions of hepsin remain unclear. Knockout of the hepsin gene in mice did not affect growth and fertility of mice, indicating that hepsin is not essential for embryogenesis. To dissect the role of hepsin in blood coagulation, this study elected to generate hepsin transgenic (Tg) mice with overexpression of hepsin in vascular endothelial system. By bringing hepsin in direct contact with plasma clotting proteins the consequences of hepsin overexpression might indicate the relation of hepsin with blood proteins. The transgenic constructs, pTHIZ-Su (wt) and pTHIZ (mut), which consist of wild-type and the active_site mutated hepsin cDNA, respectively, under the control of an endothelium specific promoter (tie2) and enhancer were verified in bovine endothelial cells by transient transfection experiments and then used for injection of the zygotes of both FVB and C57BL6/J mice. To identify cells expressing the transgene, the constructs also contain an IRES (internal ribosome entry site)-LacZ DNA as a reporter. Whole mount staining of E12.5 embryos detected LacZ expression in embryonic vascular systems. Pronucleus injections were carried out using embryos of FVB and C57BL/6 mice. Of 69 pups born to FVB mice injected with wild-type hepsin construct, Tg was detected in 34. Of these, 18 died within 24 h after birth and 16 survived till adulthood. In contrast, of 33 pups born to C57BL6/J mice injected with wild-type hepsin construct 3 with Tg died within 24 h and another 3 with Tg survived. Twenty-seven were Tg negative and all survived. Of those injected with mutant hepsin construct, Tg were detected in 11 out of 20 pups. Only 1 of the 11 Tg positive pups died within 24 h after birth. Only one of the 9 Tg negative pups died in the similar conditions. The different consequences observed in wild-type and mutant hepsin Tg mice suggest a functional role for hepsin.

目 錄
目 錄……………………………………………1
圖 目 錄……………………………………………2
表 目 錄……………………………………………3
誌 謝……………………………………………4
縮 寫 表……………………………………………6
中 文 摘 要……………………………………………7
英 文 摘 要……………………………………………9
前 言……………………………………………11
材料與方法……………………………………………17
結 果……………………………………………24
討 論……………………………………………30
圖 表……………………………………………38
參 考 書 目……………………………………………53
附 錄……………………………………………59

參 考 書 目
Asahara T. Chen D. Takahashi T. Fujikawa K. Kearney M. Magner M. Yancopoulos GD. Isner JM. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization Circulation Research. 83:233-40, 1998
Breier G. Angiogenesis in embryonic development--a review. [Review] [46 refs] Placenta. 21 Suppl A:S11-5, 2000.
Brigid H. Rosa B. Frank C. Elizabeth L. Manipulating the mouse embryo, 1994.
Brown LF. Dezube BJ. Tognazzi K. Dvorak HF. Yancopoulos GD. Expression of Tie1, Tie2, and angiopoietins 1, 2, and 4 in Kaposi's sarcoma and cutaneous angiosarcoma. American Journal of Pathology. 156:2179-83, 2000
Costantini V. Zacharski LR. Fibrin and cancer. Thromb Hasemos, 69:406-14, 1993
Drake CJ. Fleming PA. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood. 95:1671-9, 2000
Dumont DJ. Gradwohl G. Fong GH. Puri MC. Gertsenstein M. Auerbach A. Breitmen ML. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, Tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev 8424, 1897-1909.
Doumont DJ. Yamaguchi TP. Conlon RA. Rossant J. Breitman ML. Tek, a novel tyrosine kinase gene located on mose chromosome 4, is expressed in endothelial cells and their presumptive precursors. Oncogene 7,1471-148, 1992.
Dvorak HF. Dickersin GR. Dvorak AM. Manseau EJ. Pyne K. Human breast carcinoma: fibrin deposits and desmoplasia inflammatory cell type and distribution, Microvasculature and infarction. J Natl Cancer Inst 67:335-45, 1981
Fadel BM. Boutet SC. Quertermous T. Octamer-dependent in vivo expression of the endothelial cell-specific TIE2 gene. J Biol Chem 274:20376-83, 1999.
Farley D. Reymond F. Hanspeter N. Cloning and sequence analysis of rat hepsin, a cell surface proteinase. Biochimica et Biophysica Acta. 350-352, 1993
Fidler U. Tumor heterogenity and the biology of cancer invasion and metastasis Cancer Res 38:2651-60, 1978
Fujikawa K. Presman E. Isner JM. Varticovski L. Expression of tie1 and tie2 proteins during reendothelialization in balloon-injured rat carotid artery [published erratum appears in J Vasc Res 1999 Nov-Dec;36(6):509]. Journal of Vascular Research. 36:272-81, 1999.
Gordon SG. Cancer cell procoagulants and their role in malignant dsease. Semin Thromb Hasemos 18:424-33, 1992
Hongon B. Beddington R. Costantini F. Lacy E. Manipulating the mouse embryo second edition 1994.
Jaana K. Isto L. Maria H. Leena A. Juhani J. Daniel D. Kari A. Endothelial-specific Gene Expression Directed by the tie Gene Promoter In Vivo. Blood 86:1828-1835, 1995
Kawamura S. Kurachi S. Deyashiki Y. Kurachi K. Complete nucleotide sequence, origin of isoform and functional characterization of the mouse hepsin gene. Eur. J. Biochem. 262, 775-764, 1999.
Kazama Y. Hamamoto T. Foster DC. Kisiel W. Hepsin , a putative membrane-associated serine protease , activates human factor Ⅶ and initiates a pathway of blood coagulation on the cell surface leading to thrombin formation . J Biol Chem 270:66-72, 1995
Klepfish A. Greco MA. Karpatkin S. Thrombin stimulates melanoma tumor-cell binding to endothelial cells and subendothelial matrix. Int J Cancer 53:918-8223, 1993
Koblizek TI. Runting AS. Stacker SA. Wilks AF. Risau W. Deutsch U. Tie2 receptor expression and phosphorylation in cultured cells and mouse tissues. European Journal of Biochemistry. 244:774-9, 1997
Korhonen J. Polvi A. Partanen J. Alitalo K. The mouse Tie receptor tyrosine kinase gene:expression during embryonic angiogenesis. Oncogene 9, 395-403, 1994.
Kurachi K. Torres-Rosado A. Tsuji A. Hepsin. Methods in Enzymology. 244:100-14, 1994.
Lena Claesson-Welsh Vascular Growth Factors and Angiogenesis 1994
Leytus SP. Loeb KR. Hagen FS. Kurachi K. Davie EW. A Novel trypsin-like serine protease (Hepsin) with a Putative transmembrane domain expressed by human liver and hepatoma cells. Biochemistry 27:1067-74, 1998.
Lin EY. Piepkorn M. Garcia R. Byrd D. Tsou R. Isik FF. Angiogenesis and vascular growth factor receptor expression in malignant melanoma
Manfioletti G. Brancolini G. Avanzi. Schneider C. The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol 13, 4976-4985, 1993.
Nakamura T. Nishizawa M. Hagiya T. Seki M. Shimonishi A. Sugimura K. Tashiro. Shimizu S. Molecular cloning and expression of human hepatocyte griwtg factor, Nature 342, 440-443, 1989
Nierodzik MLR. Plotkin A. Kajumo F. Karpatkin S. Thrombin stimulates tumor-platelet adhesion in vitro and metsstasis in vivo. J Clin Invest 87:229-36, 1991
Oh H. Takagi H. Suzuma K. Otani A. Matsumura M. Honda Y. Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells. J Biol Chem 274:15732-9, 1999
Otani A. Takagi H. Oh H. Koyama S. Matsumura M. Honda Y. Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes. Investigative Ophthalmology & Visual Science. 40:1912-20, 1999
Patan S. TIE1 and TIE2 receptor tyrosine kinases inversely regulate embryonic angiogenesis by the mechanism of intussusceptive microvascular growth. Microvascular Research. 56:1-21, 1998
Partanen J. Dumont DJ. Functions of Tie1 and Tie2 receptor tyrosine kinases in vascular development. [Review] [67 refs] Current Topics in Microbiology & Immunology. 237:159-72, 1999.
Petersen TE. Martzen A. Ichinose. Davie EW. Characterization of thegene for human plasminogen, a key proenzyme in the fibrinolytic system. J Biol Chem 265, 6104-6111, 1990.
Puri MC. Rossant J. Alitalo K. Bernstein A. Partanen J. The receptor tyrosine kinase Tie is required for integrity and survival of vascular endothelial cells. EMBO J 14, 5884-5891, 1995
Qingyu Wu. Gene Targeting in Hemostasis. Hepsin. Frontiers in Bioscience 6, 192-200, 2001
Rao LVM. Tissur factor as a tumor procoagulant. Cancer metastasis Rev 11, 249-266, 1992.
Rapaport SI. Rao LVM. The tissue factor pathway: how it has become a “prima Ballerina”. Thromb Hasemos 1995
Rhim JS. Neoplastic transformation of human epithelial cells in vitro. Anticancer Res Sep-Oct; 9: 1345-65, 1989
Runting AS. Stacker SA. Wilks AF. tie2, a putative protein tyrosine kinase from a new class of cell surface receptor. Growth Factors. 9:99-105, 1993.
Sachs HG. McDonald TF. Membrane potentials of BHK (baby hamster kidney) cell line: ionic and metabolic determinants. J Cell Physiol Dec 80: 347-58, 1972
Schmidberger H, King L, Lasky LC, Vallera DA., Antitumor activity of L6-ricin immunotoxin against the H2981-T3 lung adenocarcinoma cell line in vitro and in vivo. Cancer Res Jun 1; 50:3249-56, 1990
Schmidt C. Bladt S. Goedecke V. Brinkmann W. Zschiesche M. Sharpe E. Gherardi BS. factor/hepatocyte growth factor is essential for liver development. Nature 373, 699-702, 1995
Sedlak BJ. Booyse FM. Bell S. Rafelson ME. Comparison of two types of endothelial cells in long term culture. Thromb Haemost 35:167-77, 1976
Zhang SL. Wendy D. Edward S. Ramsay V. Bliskovski HC. Morse III. Leki TH. William C. Vass RA. Depinho BA. Efficiency Alleles of the Pctr1 Modifier Lovcus for Plasmacytoma Susceptiblity. Mol Cell Biol Jan. 310-318, 2001.
Steven FS. Griffin MM. Biol Chem Hoppe-Seyler 369 (suppli) ,137-43. 1988
Suda T. [Function of TIE2/angiopoietin in the angiogenesis and hematopoiesis]. [Review] [17 refs] [Japanese] Rinsho Ketsueki - Japanese Journal of Clinical Hematology. 41(4):251-5, 2000
Takakura N. Huang XL. Naruse T. Hamaguchi I. Dumont DJ. Yancopoulos GD. Suda T. Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis. Immunity. 9:677-86, 1998
Tanimoto H. Yan Y. Clarke J. Korourian S. Shigemasa K. Parmley TH. Parham GP. O’BrienT J. Hepsin, a cell surface serine protease identified in hepatoma cell, is overexpressed in ovarian cancer. Cancer Res.57: 2884-2887, 1997
Thorsten M. Schlaeger SB. Joel A. Lawitts G. Teichmann WR. Urban D. Sato TN. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc. Natl. Acad. Sci. 94 3058-3063, 1997
Torres-Rosado A. O’Shea KS. Tsuji A. Chou SH. kurachi K. Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth. Proc Natl Acad Sci 90:7181-5, 1993
Tsuji A. Torees-Rosado A. Arai T. LeBeau MM. Lemons RS. Chou SH. Kurachi K. Hepsin, a cell membrane-associated protease. J Biol Chem 266:16948-53, 1991
Uehara Y. O’Minowa C. Mori K. Shiota J. Kuno T. Noda NK. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 373, 702-705, 1995.
Vu TKH. Liu RW. Haaksma C. Tomasek J. Howard E W Identification and cloning of the membrane associated serine protease, hepsin, from mouse preimplantation embryos. J Biol Chem 272: 31315-31320, 1997
Willam C. Koehne P. Jurgensen JS. Grafe M. Wagner KD. Bachmann S. Frei U. Eckardt KU. Tie2 receptor expression is stimulated by hypoxia and proinflammatory cytokines in human endothelial cells. Circulation Research 87:370-7, 2000
Wong AL.Haroon ZA. Werner S. Dewhirst MW. Greenberg CS. Peter KG. Tie2 Expression and Phosphorylation in Angiogenic and Quiescent Adult Tissues. Circulation Reserch 81: 567-574, 1997
Wong MP. Chan SY. Fu KH. Leung SY. Cheung N. Yuen ST. Chung LP. The angiopoietins, tie2 and vascular endothelial growth factor are differentially expressed in the transformation of normal lung to non-small cell lung carcinomas. Lung Cancer.
Wu Q. Yu D. Post J. Halks-Miller M. Sadler J. Morser J. Generation and characterization of mice deficient in hepsin, a hepatic transmembrane serine protease. J. Clin. Invest. 101:321-326, 1998
Yamaoka K. Masuda K. Ogawa H. Takagi K. Umemoto N. Yasuoka S. Cloning and characterization of the cDNA for human airway trypsin-like protease. J Biol Chem 273:11895-901, 1998
Yuan HT. Suri C. David N. Landon. Grorge D. Yancopoulos, Adrian S. Angiopoietin-2 Is a Site-Specific Factor in Differentiation of Mouse Renal Vasculature. J. Am Soc Nephrol, 11:1055-1066, 2000
Yu IS. Chen HJ. Lee YS E. Huang PH. Lin SR. Tsai TW. Lin SW. Mouse deficient of hepsin, a serine protease, exhibits normal embryogenesis and unchanged hepatocyte regeneration ability. Thromb Haemost, 2000
Zacharski LR. Dunwiddie C. Nutt EM. Hunt J. Memoli VA. Thromb Haemost 65:545-8, 1991
Zacharski LR. Ornstein DL. Memoli VA. Rousseau SM. Kisiel W. Expression of the factor VII activating protease, hepsin, in situ in renal cell carcinoma. Thromb haemost.79: 876-877, 1998
Zacharski LR. Wojtukiewicz MZ. Costantini V. Ornstein DL. Memoli VA. Pathways of coagulation/fibrinolysis activation in maligancy. Semin Thromb Haemost 18:104-16, 1992
Zhukov A. Hellman U. Ingelman-Sundberg M. Purification and characterization of hepsin from rat liver microsomes. Biochimica et Biophysica Acta. 1337(1):85-95, 1997
岑運倩 利用鹼性磷酸酶與hepsin融合蛋白確認與hepsin相作用之分子 國立台灣大學醫學院醫事技術學研究所碩士論文 2000
胡夢君　人類CYP11A1基因啟動子在轉殖小鼠的研究　國防醫學院生命科學研究所博士論文　1998