人類補體C1抑制劑（C1 inhibitor）屬於絲氨酸蛋白酶抑制劑家族的成員。補體C1抑制劑是生理上C1s和C1r唯一具有生理意義的抑制劑。補體C1抑制劑在典型補體路徑上扮演著必要性的角色。補體C1抑制劑為血漿活化蛋白釋放酶（plasma kallikrein）和激肽釋放酶（kinin）系統內活化factor XII的主要抑制劑中的一個。有證據證明補體C1抑制劑也可抑制凝集素（lectin）途徑中的蛋白酶、替代補體路徑中的C3b、胞漿素活化劑 (plasminogen activator) 、活化factor XI, 和活化蛋白釋放（kallikrein）。在正常的乳細胞內補體C1抑制劑基因的表達有向下調節的傾向而惡性細胞的基因表達具顯著上升。因此本研究的主要目的在於研究重組補體C1抑制劑379到478片段對於人類乳癌細胞是否有任何的影響。在此本研究中我們建構了補體C1抑制劑379到478片段於質體上。將建構好的質體轉移到大腸桿菌BL21（DE3）品系內來表現此蛋白且使用榖胱甘肽轉移酶（GST）親和層析法來純化此蛋白。使用由大腸桿菌內純化好的補體C1抑制劑來研究是否此蛋白對於人類乳癌細胞（MDA MB 435s）的增生和轉移有任何的影響。結果顯示1到100 nM濃度的重組補體C1抑制劑379到478片段在4、6或8天可抑制MDA MB 435s細胞的增生。然而，該重組蛋白的濃度上升到100 nM時，培養48小時之後才顯示出對癌細胞轉移的顯著效應。實驗結果顯示重組補體C1抑制劑379到478片段對於MDA MB 435s細胞的增生和轉移均有一定的抑制效果。

Human C1 inhibitor (C1 INH) belongs to the superfamily of serine protease inhibitors (serpins). It is the only known physiological inhibitor of C1s and C1r, therefore C1 INH plays an essential role as a regulator of complement classical pathway. C1 INH is also one of the major inhibitors of plasma kallikrein and activated factor XII of the kinin system. There is evidence that C1 INH can also inhibit the complement lectin pathway proteases, and the complement alternative pathway (C3b), plasminogen activator, activated factor XI, and kallikrein. A recent report indicated C1 inhibitor was significantly down-regulated in normal breast cells compared to malignant cell line. Thus, the major aim of this project is to study the effect of recombinant C1 inhibitor Ile379-Ala478 on human breast cancer cells. In this study, we constructed plasmid containing DNA fragment for C1 inhibitor C-terminal negion of Ile379-Ala478. Then the constructed plasmid was transferred into E.coli BL21(DE3) for expression. The recombinant protein was purifed by GST-affinity chromatography. Recombinant C1 inhibitor produced by E.coli was used to treat breast cancer cells (MDA MB 435s) to study its effect on cell proliferation and migration. Results showed that recombinant C1 inhibitor Ile379-Ala478 protein inhibited proliferation of MDA MB 435s cells after four, six or eight days, incubation in the presence of recombinant protein (1~100 nM). However, cell migration was inhibited only at 100 nM on 48 hrurs incubation..Our experimental data showed that the recombinant C1 inhibitor Ile379-Ala478 inhibited proliferation and migration of MDA MB 435s cells.

Contents
Abstract •••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1
Abstract (in Chinese) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2
Abbreviation • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •3
Chapter 1 Introduction• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •4
Chapter 2 Materials and methods•••• • • • • • • • • • • • • • • • • • • • • • • • • 14
2.1 Materials••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 14
2.2 Approaches in molecular biology • • • • • • • • • • • • • • • • • • • • • • • • 17
2.2.1 Preparation of template DNA fragment for PCR • • • • • • • • • • • • • 17
2.2.2 Amplification of C1 inhibitor Ile379-Ala478 gene by PCR•• • • • • •••17
2.2.3 T-A cloning ••••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •18
2.2.4 Transformation of intermediate plasmid••• • • • • • • • • • • • • • • • • •19
2.2.5 Purification of plasmids and checking••••••• • • • • • • • • • • • • • • • •20
2.2.6 Ligation of pGEX-2T and C1 inhibitor Ile379-Ala478••••• • • • • • • 20
2.2.7 Checking the plasmid of pGEX-2T and C1 inhibitor Ile379-Ala478•21
2.3 Approaches in recombination protein detection ••• • • • • • • • • • • • • •21
2.3.1 Expression and purification of C1 inhibitor Ile379-Ala478 ••••• • • •21
2.3.2 Sodium dodecylsulfate-polyacryamide gel electrophoresis ••• • • • • 22
2.4 Approaches in cell biology •• • • • • • • • • • • • • • • • • • • • • • • • • • • •22
2.4.1 Cells and culture conditions ••• • • • • • • • • • • • • • • • • • • • • • • • • 22
2.4.2 In vitro cell proliferation assay• • • • • • • • • • • • • • • • • • • • • • • • •22
2.4.3 In vitro wound healing assay •••••• • • • • • • • • • • • • • • • • • • • • • •23
Chapter 3 Results••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 25
3.1 Polymerase chain reaction amplification and PCR cloning •••• • • • • •25
3.2 Expression and purification of C1 inhibitor Ile379-Ala478 •• • • • • • •25
3.3 In vitro cell proliferation assay •• • • • • • • • • • • • • • • • • • • • • • • • • •26
3.4 In vitro wound healing assay• • • • • • • • • • • • • • • • • • • • • • • • • • • • 27
Chapter 4 Discussion • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 29
Reference • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •33

Alsenz, J., K. Bork, and M. Loos. 1987. Autoantibody-mediated acquired deficiency of C1 inhibitor. N Engl J Med 316:1360-1366.
Baker, M. S, P. Bleakley, G. C. Woodrow, and W. F. Doe. 1990. Inhibition of cancer cell urokinase plasminogen activator by its specific inhibitor PAI-2 and subsequent effects on extracellular matrix degradation.Cancer Res 50(15):4676-4684.
Beinrohr, L., V. Harmat, J. Dobo, L. Zsolt, P. Gal, and P. Zavodszky. 2007. C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease. J Biol Chem 282:21100-21209.
Bensa, J. C., A. Reboul, and M. G. Colomb. 1983. Biosynthesis in vitro of complement subcomponents C1q, C1s and C1 inhibitor by resting and stimulated human monocytes. Biochem J 216(2):385-392.
Bergamaschini, L., G. Gobbo, S. Gatti, L. Caccamo, P. Prato, M. Maggioni, P. Braidotti, R. Di Stefano, and L. R. Fassati. 2001. Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver. Clin Exp Immunol 126:412-420.
Bock, S. C., K. Skriver, E. Nielsen, H. C. Thøgersen, B. Wiman, V. H. Donaldson, R. L. Eddy, J. Marrinan, E. Radziejewska, and R. Huber. 1986. Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization. Biochemistry 25:4292-4301.
Cai, S., and A. E. Davis. 2003. Complement regulatory protein C1 inhibitor binds to selectins and interferes with endothelial-leukocyte adhesion. J Immunol 171:4786-4791.
Cai, S., V. Dole, W. Bergmeier, J. Scafidi, H. Feng, D. D. Wagner, and A. E. Davis. 2005. A direct role for C1 inhibitor in leukocyte adhesion. J Immunol 174:6462-6466.
Cajot, J. F., J. Bamat, G. E. Bergonzelli, E. K. Kruithof, R. L. Medcalf, J. Testuz, and B. Sordat. 1990. Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells. Proc Natl Acad Sci U S A 87(18):6939-6943.
Carter, P. E., C. Duponchel, M. Tosi ,and J. E. Fothergill. 1991. Complete nucleotide sequence of the gene for human C1 inhibitor with an unusually high density of Alu elements. Eur J Biochem 197:301-308.
Castelli, R., D. L. Deliliers, L. C. Zingale, E. M. Pogliani, and M. Cicardi. 2007. Lymphoproliferative disease and acquired C1 inhibitor deficiency. Haematologica 92:716-718.
Cicardi, M., G. Bisiani, M. Cungo, P. Spaeth, and A. Agostoni. 1993. Autoimmune C1-inhibitor deficiency. Report of eight patients. Am J Med
95:169-175.
Cohen, S. H., S. M. Koethe, F. Kozin, G. Rodey, J. A. Arkins, and J. N. Fink. 1978. Acquired angioedema associated with retal carcinoma and its response to danazol therapy. J Immunol 62:217-212.
Colten, H. R. 1972. Ontogeny of the Human Complement System: In Vitro Biosynthesis of Individual Complement Components by Fetal Tissues. J Cli Invest 51:725-730.
Coutinho, M., K. S. Aulak, and A. E. Davis 3rd. 1994. Functional analysis of the serpin domain of C1 inhibitor. J Immunol 153:3648-3654.
Donaldson, V. H. 1993. C1-inhibitor and its genetic alterations in hereditary angioneurotic edema. Int Rev Immunol 10:1-16.
Farkas, H., A. Csepregi, and E. Nemesanszky. 1999. Acquired angioedema associated with chronic hepatitis C. J Immunol 103:711-712.
Farkas, H., M. Szongoth, and M. Bely. 2001. Angioedema due to acquired deficiency of C1-esterase inhibitor associated with leucocytoclastic vascilitis. Acta Derm Venereol 81:298-300.
Folkerd, E. J., B. Gardner, and N. C. Hughes-Jones. 1980. The relationsip between the binding ability and the rate of activation of the complement component C1. Immunol 41:179-185.
Gadek, J. E., S. W. Hosea, J. A. Gelfand, M. Santaella. 1980. Replacement therapy in hereditary angioedema: successful treatment of acute episodes of angioedema with partly purified C1 inhibitor.N Engl J Med 302:542-546.
Gulati, P., C. Lemercier, D. Guc, D. Lappin, and W. Whaley. 1993. Regulation of the synthesis of C1 subcomponents and C1-inhibitor. Behring Inst Mitt 93:196-203.
Harrison, R. A. 1983. Human C1 inhibitor: improved isolation and preliminary structural characterization. Biochemistry 22:5001-5007.
He, S., R. B. Sim, and K. Whaley. 1996. Epitope mapping of C1 inhibitor autoantibodies from patients with acquired C1 inhibitor deficiency.J Immunol 156(5):2009-2013.
He, S., R. B. Sim, and K. Whaley. 1997a. A secondary C1s interaction site on C1-inhibitor is essential for formation of a stable enzyme-inhibitor complex. FEBS Lett 405:42-46.
He, S., J. C. Yang, S. Tsang, R. B. Sim and K. Whaley. 1997b. Role of the distal hinge region of C1-inhibitor in the regulation of C1s activity. FEBS Lett 412:506-510.
He, S., R. B. Sim, and K. Whaley. 1998. Mechanism of Action of Anti-C1-Inhibitor Autoantibodies: Prevention of the Formation of Stable C1s-C1-inh Complexes. Med 4:119-128.
He, S., and R. A. Stockley. 2002. SDS-stable C1 inhibitor-C1s complex is linked by an oxygen bridge: Implication for the formation of stable serpin- enzyme ccomplexes. Recent Res Devel Immunology 4: 803-809.
Iatridis, S. G., and J. H. Ferguson. 1962. Actie Hageman factor: a plasma lysokinase of the human fibrinolytic system. J Clin Invest 41:1277-1287.
Jiang, H., E. Wagner, and M. M. Frank. 2001. Complement 1 inhibitor is regulator of the alternative complement pathway. J Exp Med 1609-1616.
Kaplan, A. P., and K. F. Austen. 1970. A pre-albumin activator of prekallikrein. J Immunol 105(4):802-811.
Kaplan, A. P., and K. F. Austen. 1971. A prealbumin activator of prekallikrein: II.Derivation of activators of prekallikrein from active Hageman factor by digestion with plasmin. J Exp Med 133(4):696-712.
Kaplan, A. P., and K. F. Austen. 1972. The fibrinolytic pathway of human plasma. Isolation and characterization of the plasminogen proactivator. J Exp Med 135(6):1378-1393.
Katz, Y., and R. C. Strunk. 1989. Synthesis and regulation of C1 inhibitor in human skin fibroblasts. J Immunol 142(6):2041-2045.
Lappin, D. F., G. D. Birnie, and K. Whaley. 1990. Modulation by interferons of the expression of monocyte complement genes. Biochem J 268:387-392
Leibovitz, A. 1963. The growth and maintainance of tissue/cell cultures in free gas exchange with the atmosphere. Amer J Hyg 78:173-180
Levy, N. J., N. Ramesh, M. Cicardi, R. A. Harrison, and A. E. Davis III. 1990. Type II hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine436 in the C1 inhibitor gene. Proc Natl Acad Sci U S A 87:265-268.
Liang, C., A. Y. Park, and J. Guan. 2007. In vitro scratch assay: a conventient and inexpensive method for analysis of cell migration in vitro. Nature Protocols 2:329-333.
Liang, X., J. Huuskonen, M. Hajivandi, and R. Manzanedo. 2009. Identification and quantification of proteins differentially secreated by a pair of normal and malignant breast-cancer cell lines. Proteomics 182-193.
Lin,Y. L.,and S. He. 2006. Sm22.6 antigen is an inhibitor to human thrombin. Molecular&Biochemical Parasitology 147:95-100.
Liu, D., S. cai, Gu. X, J. Scafidi, Wu. X, and A. E. Davis III. 2003. C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide. J Immunol 171:2594-2601.
Liu, D., C. C. Cramer, J. Scafidi, and A. E. Davis III. 2005. N-linked glycosylation at Asn3 and the positively charged residue within the amino-terminal domain of the c1 inhibitor are required for interaction of the C1 Inhibitor with Salmonella enterica serovar typhimurium lipopolysaccharide and lipid A. Infect Immun 73:4478-4487.
Lonn, A., M. Garfonyi, W. Van Zyl, B. Hahn-Hägerdal, and R. C. Otero.
2002. Cold adaptation of xylose isomerase from Thermus thermophilus
through random PCR mutagenesis. Gene cloning and protein
characterization. Eur J Biochem 269(1):157-163.
Margolis, J. 1958. Activation of a permeability factor in plasma by contact with glass. Nature 181:635-636.
Miao, R. Q, J. Agata, L. Chao, and J. Chao. 2002. Kallistatin is a new inhibitor of angiogenesis and tumor growth. Blood 100(9):3245-3252.
Mogens. H. N., B. Soren, A. N. Jens, and H. C. Mogens. 1998. C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro. I Immunology 10(2):167-173.
Ogston, D., C. M. Ogston, O. D. Ratnoff, and C. D. Forbes. 1969. Studies
on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation of Hageman factor and additional cofactors. J Clin Invest 48(10):1786-801.
Oltvai, Z. N., E. C. C. Wong, J. P. Atkinson, and K. S. K. Tung. 1991. C1-inhibitor deficiency:molecular and immunological basis of hereditary and acquired angioedema. Lab Invest 65:381-388.
Perkins, S. J., K. F. Smith, S. Amatayakul, D. Ashford, T. W. Rademacher, R. A. Dwek, P.J. Lachmann, and R. A. Harrison. 1990. Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering. J Mol Biol 214: 751-763.
Ratnoff, O. D., and I. Lepow. 1957. Some properties of an esterase derived from preparations of the first component of complement. J Exp Med 106:327-343.
Ratnoff, O. D., E. W. Davie, and D. L. Mallett. 1961. Studies on the action of Hageman factor: evidence that activated Hageman factor in turn activates plasma thromboplastin antecedent. J Clin Inves 40:803-819.
Robles, J. and M. Doers. (1994) pGEM®-T Vector Systems troubleshooting guide. Promega Notes 45:19-20.
Salvesen, G. S., J. J. Catanese, L. F. Kress, and J. Travis. 1985. Primary structure of the reactive site of human C1-inhibitor. J Biol Chem 260: 2432-2436.
Schapira, M., C. F. Scott, and R. W. Colman. 1982. Contribution of plasma protease inhibitots to the inactivation of kallikrein in plasma. J Clin Invest 462-468.
Sim, R. B., A. Reboul, G. J. Arlaud, C. L. Villiers, and M. G. Colomb. 1979. Interation of 125I-labelled complement subcomponents C1r and C1s with protease inhibitors in plasma. FEBS Lett 97:111-115.
Skriver, K., W. R. Wikoff, P. A. Patston, F. Tausk, M. Schapira, A. P. Kaplan, and S. C.Bock . 1991. Substrate properties of C1 inhibitor Ma (alanine 434-glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status. J Biol Chem 266(14):9216-9221.
Smith, D. B., K. S. Johnson. 1988. Single-step purification of
polypeptides expressed in Escherichia coli as fusions with glutathione
S-transferase. Gene 67 (1):31-40.
Stoppa-Lyonnet, D., C. Duponchel, T. Meo, J. Laurent, P. E. Carter, M. Arala-Chaves, J. H. Cohen, G. Dewald, J. Goetz, G. Hauptmann. 1991. Recombinational biases in the rearranged C1-inhibitor genes of hereditary
angioedema patients. Am J Hum Genet 49:1055-1062.
Webster, M. E, and O. D. Atnoff. 1961. Role of Hageman factor in the activation of vasodilator activity in human plasma. Nature 192:180-181.
Vapnek, D., N. K. Alton, C. L. Bassett, and S. R. Kushner. 1976. Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I. Proc Natl Acad Sci U S A 73(10):3492-3496.
Xiao, G., Y. E. Liu, R. Gentz, and Y. E. Shi. 1999. Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary myoepithelial cells.
Ziccardi, R. J. 1981. Activation of the early components of the classical complement pathway under physiological condictions. J Immunol 1768-1773.
Zingale, L. C., R. Castelli, A. Zanichelli, M. Cicardi. 2006. Acquired deficiency of the inhibitor of the first complement component: presentation, diagnosis, course, and conventional management. Immunol Allergy Clin North Am 26:669-690.
何世屏，林雅玲，凱斯٠威利(1995年)第一補體抑制蛋白的醣基化多肽的穩定和功能是必需的。中國科學技術(生物部份),347-354
廖彩岑(2008年) SjGST蛋白對人類乳癌細胞的生長及遷移之影響。國立中山大學生物科學系碩士論文