臺灣博碩士論文加值系統

感染A 群鏈球菌會引起相當廣泛的疾病，從輕微自癒性的，到嚴重危及生命的感染，如咽喉炎、扁桃腺炎、中耳炎和小膿皰疹等輕微的症狀到壞疽性肌膜炎、菌血症和鏈球菌毒素休克症候群等嚴重疾病。臨床上疾病嚴重程度的不同，除了可能感染菌株血清型不同以外，病人本身的先天體質條件也可能影響。在之前研究中發現敗血症病人的血液中kallistatin 蛋白質量呈現下降的現象，在當時推測是因為有需要kallistatin 作用才消耗而減少，但是真正的關係並不清楚。Kallistatin 原本是kallikrein 結合蛋白，但是現在已陸續發現kallistatin 可以不透過和kallikrein 結合而執行功能，例如kallistatin 的抗血管新生、抗發炎和抗氧化等功能。到目前為止，只知道kallistatin 與高血壓、心血管疾病和腎臟病等慢性疾病有相關，對於微生物感染，kallistatin 並沒有被探討過。因此，本論文探討kallistatin 在A 群鏈球菌感染的小鼠模式中可能扮演的角色。我們初步以hydrodynamic 注射方式讓小鼠短暫而大量的表現kallistatin 之後進行皮下氣囊感染A 群鏈球菌，發現小鼠的存活率明顯提升，局部感染的細菌量和菌血症的情形減少很多。在血漿和局部感染的氣囊回抽液中，也發現發炎的細胞激素和趨化
因子都有降低的情形。然而和控制組比較之下，表現kallistatin 的小鼠中，在受感染部位所浸潤的嗜中性白血球數量較少，但是這些浸潤的免疫細胞卻有較高的存活率。另外，我們以組織切片直接觀察，發現不管是感染的皮膚還是肝臟組織，只要是在表現kallistatin 組別中，受損的情形都比較輕微。總結以上研究，我們知道kallistatin 對於A 群鏈球菌感染小鼠具有抗發炎的保護效果，但是kallistatin在感染過程中所參與的作用機制和是否在感染後具有治療效果，還需要進一步研究。

The group A streptococcus (GAS) causes a variety of diseases, ranging from mild self-limiting infection to severe life-threatening infection, such as pharyngitis, tonsillitis, and tympanitis to necrotizing fasciitis, bacteremia, and streptococcus toxic shock syndrome (STSS). The disease severity may be associated with the different bacterial serotype or depend on patient’s healthy condition. Previous clinical findings indicated that patients with sepsis had lower level of kallistatin protein. Kallistatin was originally found to be a tissue kallikrein-binding protein. But recent studies have shown that kallistatin may function independently of its interaction with tissue kallikrein. Kallistatin has some biological effects, such as anti-angiogenesis, anti-inflammation, and anti-oxidation and possesses beneficial effects in hypertensive, cardiovascular and renal diseases. However, the effects of kallistatin in microbial infection have not been explored. In this study, we transiently overexpressed kallistatin gene by hydrodynamic injection and investigated the protective effects of kallistatin in a mouse model of GAS infection. We found that kallistatin increased the survival rate of GAS-infected mice. The bacterial numbers and the inflammatory cytokines and chemokines in the local infection site and blood were lower in kallistatin-treated group than those in control groups. In contrast with control mice, kallistatin-treatment decreased neutrophil infiltration into the local infection site, while promoting neutrophil viability. In the histological analysis, the skin and liver tissue showed less damage after treatment with kallistatin compared to the control groups. Consequently, we will examine the biological function and therapeutic effect of kallistatin in GAS-infected mice.

中文摘要 I
英文摘要 II
致謝 III
目錄 IV
表目錄 VI
圖目錄 VII
縮寫檢索表 X

緒論 1
特定目標與實驗設計 6
材料
1. 實驗動物 7
2. 細菌株 7
3. Kallistatin基因載體 7
4. 試劑 7
5. 耗材 8
6. 儀器 9
方法
1. Kallistatin基因載體製備 11
2. 重組蛋白kallistatin基因表現的小鼠模式 11
3. A群鏈球菌之感染小鼠模式 12
4. 表現kallistatin之小鼠感染A群鏈球菌後外觀病徵及致死率觀察 12
5. 感染後血液及感染部位氣囊回抽液的收集 12
6. 血液與氣囊回抽液內菌量計數 13
7. 感染部位免疫細胞浸潤分析 13
8. 血漿與氣囊回抽液的kallistatin蛋白，cytokine和chemokine測定 13
9. 感染部位與器官的病理變化 14
10. 血漿生化分析 14
11. 統計 14
結果 1. 重組蛋白kallistatin基因表現的小鼠模式 15
2. A群鏈球菌之小鼠感染模式與kallistatin表現的有無對小鼠致死率的影響 15
3. 感染後小鼠仍然持續表現kallistatin蛋白 16
4. 感染部位皮膚潰爛滲血面積大小 16
5. 感染部位氣囊回抽液的細菌數 17
6. 感染A 群鏈球菌引發的嗜中性白血球浸潤 17
7. 感染部位氣囊皮膚組織切片染色 18
8. 感染後菌血症情形 18
9. 感染部位氣囊回抽液和血漿中細胞激素和趨化因子表現量 19
10. 感染後器官的組織病理變化 19
11. 感染後肝發炎指數 20
12. Kallistatin對A群鏈球菌感染之小鼠的治療效果 20

討論
1. 重組蛋白kallistatin基因表現的小鼠模式 22
2. A群鏈球菌之小鼠感染模式與kallistatin表現的有無對小鼠致死率的影響 24
3. Kallistatin 在感染A群鏈球菌的小鼠模式中可能的生物角色 24
4. Kallistatin表現模式在A群鏈球菌感染後對肝臟的影響 27
5. Kallistatin對感染A群鏈球菌小鼠之治療效果

參考文獻 29
表 33
圖 34
附錄一 49
附錄二 50
自述 51

Bisno, A. L., Brio, M. O., and Collins, C. M. Molecular basis of group A streptococcal virulence. Lancet Infect. Dis. 3:191-200, 2003.
Batzloff, M. R., Pandey, M., Olive, C., and Good, M. F. Advances in potential M-protein peptide-based vaccines for preventing rheumatic fever and rheumatic heart disease. Immunol. Res. 35:233-248, Review 2006.
Baker, H. M., Proft, T., Webb, P. D., Arcus, V. L., Fraser, J. D., and Baker, E. N. Crystallographic and mutational data show that the streptococcal pyrogenic exotoxin J can use a common binding surface for T-cell receptor binding and dimerization. J. Biol. Chem. 10;279:38571-38576, 2004.
Banerjee, A., Chisti, Y., and Banerjee, U. C. Streptokinase--a clinically useful thrombolytic agent. Biotechnol. Adv. 22:287-307, Review 2004.
Chao, J., Schmaier, A., Chen, L. M., Yang, Z., and Chao, L. Kallistatin, a novel human tissue kallikrein inhibitor: levels in body fluids, blood cells, and tissues in health and disease. J. Lab. Clin. Med. 127:612-620, 1996.
Chao, J., Yin, H., Yao, Y. Y., Shen, B., Smith, R. S., and Chao, L. Novel role of kallistatin in protection against myocardial ischemia-reperfusion injury by preventing apoptosis and inflammation. Hum. Gene Ther. 17:1201-1213, 2006.
Chen, L. M., Chao, L., and Chao, J. Beneficial effects of kallikrein-binding protein in transgenic mice during endotoxic shock. Life Sci. 60:1431-1435, 1997.
Cleary, P. P., Prahbu, U., Dale, J. B., Wexler, D. E., and Handley, J. Streptococcal C5a peptidase is a highly specific endopeptidase. Infect. Immun. 60:5219-5223, 1992.
Collin, M., and Olsen, A. Extracellular enzymes with immunomodulating activities: variations on a theme in Streptococcus pyogenes. Infect. Immun. 71:2983-2992, Review 2003.
Dale, J. B., Washburn, R. G., Marques, M. B., and Wessels, M. R. Hyaluronate capsule and surface M protein in resistancee to oponization of group A streptococci. Infect. Immun. 64:1495-1501, 1996.
D’Costa, S. S., and Boyle, M. D. Interaction of group A streptococcus within human plasma results in assembly of a surface plasminogen activator that contributes to occupancy of surface plasmin-binding structures. Microb. Pathog. 24:341-349, 1998.
DeAngelis, P. L., Yang, N., and Weigel, P. H. The Streptococcus pyogenes hyaluronidase synthetase: sequence comparison and conservation among various group A strains. Biochem. Biophy. Res. Commun. 199:1-10, 1994.
Facklam, R. F., Martin, D. R., and Lovgren, M. Extension of the Lancefield classification for group A streptococci by addition of 22 new M protein gene sequence types from clinical isolate: emm103 to emm124. Clin. Infect. Dis. 34:28-38, 2002.
Feito, M. J., Sanchez, A., Oliver, M. A., Perez-Caballero, D., Rodriguez de Cordoba, S., Alberti, S., and Rojo, J. M. Membrane cofactor protein (MCP, CD46) binding to clinical isolates of Streptococcus pyogenes: Binding to M type 18 strains is independent of Emm or Enn proteins. Mol. Immunol. 44:3571-3579, 2007.
Guilherme, L., Kalil, J., and Cunningham, M. Molecular mimicry in the autoimmune pathogenesis of rheumatic heart disease. Autoimmunity 39:31-39, Review 2006.
Guilherme, L., Fae, K. C., Oshiro, S. E., Tanaka, A. C., Pomerantzeff, P. M., and Kalil, J. T cell response in rheumatic fever: crossreactivity between streptococcal M protein peptides and heart tissue proteins. Curr. Protein Pept. Sci. 8:39-44, Review 2007.
Hauser, A. R., Stevens, D. L., Kaplan, E. L., and Schlievert, P. M. Molecular snslysis of pyogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome. J. Clin. Microbiol. 29:1562-1567, 1991.
Ji, Y., McLandsboroigh, L., Kondagunta, A., and Cleary, P. P. C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect. Immun. 64:503-510, 1996.
Kuo, C. F., Wu, J. J., Lin, K. Y., Tsai, P. J., Lee, S. C., Jin, Y. T., Lei, H. Y., and Lin, Y. S. Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection. Infect. Immun. 66:3931-3935, 1998.
Kuo, C. F., Wu, J. J., Tsai, P. J., Kao, F. J., Lei, H. Y., Lin, M. T., and Lin, Y. S. Streptococcal pyrogenic exotoxin B induces apoptosis and reduces phagocytic activity in U937 cells. Infect. Immun. 67:126-130, 1999.
Kuo, C. F., Luo, Y. H., Lin, H. Y., Huang, K. J., Wu, J. J., Lei, H. Y., Lin, M. T., Chuang, W. J., Liu, C. C., Jin, Y. T., Lin, Y. S. Histopathologic changes in kidney and liver correlate with streptococcal pyrogenic exotoxin B production in the mouse model of group A streptococcal infection. Microb Pathog. 36:273-85, 2004
Keith, J. C. Jr., Sainz, I. M., Isordia-Salas, I., Pixley, R. A., Leathurby, Y., Albert, L. M., and Colman, R. W. A monoclonal antibody against kininogen reduces inflammation in the HLA-B27 transgenic rat. Arthritis Res. Ther. 7:R769-776, 2005.
Liu, F., Song, Y. K., and Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Thera. 6:1258-1266, 1999.
Madden, J. C., Ruiz, N., and Caparon, M. Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in gram-positive bacteria. Cell 104:143-152, 2001.
Miao, R. Q., Murakami, H., Song, Q., Chao, L., and Chao, J. Kallistatin stimulates vascular smooth mouscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery. Circ. Res. 86:418-424, 2000.
Miao, R. Q., Agata, J., Chao, L., and Chao, J. Kallistatin is a new inhibitor of angiogenesis and tumor growth. Blood 100:3245-3252, 2002.
Musser, J. M., Hauser, A. R., Kim, M. H., Schlievert, P. M., Nelson, K., and Selander, R. K. Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive disease: clonal diversity and pyogenic exotoxin expression. Proc. Natl. Acad. Sci. USA 88:2668-2672, 1991.
Ohara-Nemoto, Y., Sasaki, M., Kaneko, M., Nemoto, T., and Ota, M. Cysteine protease activity of streptococcal pyrogenic exotoxin B. Can. J. Microbiol. 40:930-936, 1994.
Olive, C. Progress in M-protein-based subunit vaccines to prevent rheumatic fever and rheumatic heart disease. Curr. Opin. Mol. Ther. 9:25-34, Review 2007.
Purushothaman, S. S., Park, H. S., and Cleary, P. P. Promotion of fibronectin independent invasion by C5a peptidase into epithelial cells in group A Streptococcus. Indian. J. Med. Res. 119:44-47, 2004.
Proft, T., Webb, P. D., Handley, V., and Fraser, J. D. Two novel superantigens found in both group A and group C Streptococcus. Infect. Immun. 71:1361-1369, 2003.
Papageorious, A. C., and Acharya, K. R. Microbial superantigens: form structure to function. Trends. Microbil. 8:369-357, 2000.
Robinson, J. H., and Kehoe, M. A. Group A streptococcal M proteins: virulence factors and protective antigens. Immunol. Today 13:362-367, 1992.
Starr, C. R., and Engleberg, N. C. Role of hyaluronidase in subcutaneous spread and growth of group A streptococcus. Infect. Immun. 74:40-48, 2006.
Sachse, S., Seidel, P., Gerlach, D., Gunther, E., Rodel, J., Straube, E., and Schmidt, K. H. Superantigen-like gene(s) in human pathogenic Streptococcus dysgalactiae, subsp equisimilis: genomic localisation of the gene encoding streptococcal pyrogenic exotoxin G (speG(dys)). FEMS Immunol. Med. Microbiol. 11;34:159-167, 2002.
Stevens, D. L. Invasive group A streptococcus infections. Clin. Infect. Dis. 14:2-13, 1992.
Tewodros, W., and Kronvall, G. M protein gene (emm type) analysis of Group A Beta-hemolytic Streptococci from Ethiopia reveals unique patterns. J. Clin. Microbiol. 43:4367-4379, 2005.
Tsai, P. J., Lin, Y. S., Kuo, C. F., Lei, H. Y., and Wu, J. J. Group A Streptococcus induces apoptosis in human epithelial cells. Infect. Immun. 67:4334-4339, 1999.
Wang, C. R., Chen, S. Y., Wu, C. L., Liu, M. F., Jin, Y. T., Chso, L., and Chao, J. Prophylactic adenovirus-mediate human kallistatin gene therapy suppresses rat arthritis by inhibiting angiogenesis and inflammation. Arthritis Rheum. 52:1319-1324, 2005.
Zhang, G., Gao, X., Song, Y. K., Vollmer, R., Stolz, D. B., Gasiorowski, J. Z., Dean, D. A., and Liu, D. Hydroporation as the mechanism of hydrodynamic delivery. Gene Thera. 11:675-682, 2004.
行政院衛生署疫情報導 “法定傳染病統計結果” 2006.
羅月霞 “Correlation of SPE B with histopathologic change, NO, and cytokine production in a mouse model of Group A streptococcal infection” 國立成功大學微生物免疫學研究所碩士論文. 2001.
吳岳穎 “ Streptococcal pyogenic Exotoxin B-mediated Inhibition of Apoptotic cell clearance by Macrophages” 國立成功大學微生物免疫學研究所碩士論文. 2004.