摘要
A群鏈球菌(group A streptococci)的感染會導致多種疾病，臨床上所產生的嚴重併發症包括壞疽性肌膜炎(necrotizing fasciitis)及鏈球菌毒性休克症候群(streptococcal toxic shock syndrome)。但是到目前為止它們的致病因子還不甚了解。越來越多的證據顯示，熱原性外毒素B (SPE B)在侵襲性A群鏈球菌感染時是重要的毒力因子。SPE B的基因存在於所有A群鏈球菌中，它產生一種屬於cysteine protease的蛋白脢。為了直接探討SPE B在A群鏈球菌感染時擔任的角色，先前已利用基因重組技術得到不能產生蛋白脢的speB isogenic mutants。因為很多嚴重A群鏈球菌的感染病例都是由皮膚及軟組織受到感染開始，所以我們實驗室就以皮下氣囊感染的方式來研究SPE B在A群鏈球菌感染小鼠時扮演的角色，結果發現這些突變株對小鼠的致死率及局部組織的傷害都遠低於其野生株。而且，利用SPE B免疫的方式可保護小鼠接受致死菌量的野生株感染﹔這樣的結果更加證明了SPE B的角色。為了解A群鏈球菌感染病程的發展和SPE B的關係，本論文則更進一步去探究SPE B在局部A群鏈球菌感染時對全身性組織病理變化及此菌深部侵襲力的影響。計數起始感染部位的菌數，發現受野生菌株感染小鼠之氣囊回抽液的菌數會隨著時間增加，但感染突變株的則不會。感染野生株的小鼠發展為菌血症的機率遠高於感染突變株的。對多種器官做病理組織檢查，發現肝臟及腎臟有嚴重的組織破壞。肝及腎的病變在小鼠接種細菌後24小時就可見到，而且感染野生株後組織的傷害遠大於感染突變株後的。血清生化成份(AST﹑ALT及BUN)的量也隨著肝及腎的病變而上升，而且感染野生株小鼠血清中的量也遠大於感染突變株小鼠的。腦及肺組織並沒有出現病變。感染野生株小鼠脾臟的重量在48小時明顯下降，但感染突變株的則不會。我們也利用競爭性ELISA測得菌接種部位和血清中SPE B的上升。除此之外，我們利用recombinant wild-type SPE B及它的mutant C192S分別直接以腹腔注射的方式給予小鼠，結果發現wild-type SPE B會引起小鼠局部組織的損壞及小鼠的死亡，而C192S則不會。總之，SPE B在A群鏈球菌感染小鼠的模式中﹐對細菌分布及器官組織的影響﹐在本論文中已被證實。

Abstract
Group A streptococci (GAS) streptococcus pyogenes causes a broad spectrum of diseases. Clinical features that result in life-threatening sequelae include necrotizing fasciitis and streptococcal toxic shock syndrome. However, so far, their pathogenic factors are poorly defined. Accumulated evidence suggests that streptococcal pyrogenic exotoxin B (SPE B) may be a critical virulent factor in invasive GAS infections. The gene of SPE B is found in all strains of GAS, and the encoded protein functions as a cysteine protease. In order to study the role that SPE B may play in the pathogenesis of GAS infection, the isogenic protease-negative mutants were previously constructed by homologous recombination. The common primary focuses of the severe GAS infections are infections of the skin and soft tissue (necrotizing fasciitis or myositis). We have adopted skin air sac as the route of bacterial infection for studying the role of SPE B in the mouse model of GAS infection. Previous studies in our laboratory indicated that these mutants caused less mortality and tissue damage than protease-positive strains. The role of SPE B was further confirmed by demonstrating that SPE B immunization of mice conferred protection from challenge with a lethal dose of protease-positive bacteria. To further explore the correlation of SPE B with the process of GAS diseases, the systemic effects by local infection were investigated in this study. We found that bacterial numbers in the exudates from the air pouches of mice infected with the wild-type strains were increased by the time, but not seen in mice infected with the speB mutants. The frequency of mice infected with the wild-type strains developed bacteremia was higher than that of mice infected with the speB mutants. Further, to observe whether the multiple organs were affected, the histopathologic changes of various organs were examined. We found severe tissue destruction in kidney and liver. Histopathologic changes in kidney and liver occurred at 24 h after bacteria inoculation, and the wild-type strains caused a higher severity compared to those of the speB mutants. The elevation of biochemical components (AST﹑ALT and BUN) levels in sera was correlated with liver and renal impairment, and the levels were higher in sera from mice infected with the wild-type strains than those from mice infected with the speB mutants. The histopathologic changes were not observed in brain and lung. The organ weights of spleen from mice infected with the wild-type strains were reduced at 48 h, but not seen in mice infected with the speB mutants. The increase of SPE B level in inoculation site and serum was detected with competition ELISA. In addition, the direct effects of SPE B in mice via intraperitoneal injection were studied using the recombinant wild-type SPE B and its mutant C192S. The results indicated that wild-type SPE B but not C192S caused local tissue damage and death in the animal. Taken together, a correlation of SPE B with bacteriologic and histopathologic changes is demonstrated in this study.

目錄
中文摘要……………………………………………………. I
英文摘要………………………………………………….. III
誌謝………………………………………………………... V
目錄……………………………………………………….. VI
圖目錄……………………………………………………… X
表目錄………………………………………………… …. XII
符號及縮寫………………………………………………. XIII
主文
緒論………………………………………………………… 1
材料與方法
I、 材料………………………………………………….… 9
A、實驗動物 ……………………………………………... 9
B、細菌株 …………………………………………….….. 9
C、 SPE B之抗體………………………………………... 10
D、重組的SPE B ……………………………………….. 10
II、方法
A、A群鏈球菌之小鼠感染模式…………………………… 11
1) 起始感染部位菌量的計數……………………………. 11
2) 器官的病理變化………………………………………. 11
3) 肝臟組織切片的特殊染色(Sudan III) ……………….. 12
4) 血液及器官的菌量計數及稱重………………………. 12
5) 血清中生化成份的測定………………………………. 13
B、 競爭性酵素聯結免疫分析法…………………………... 13
C、 小鼠胸腺細胞數目的測定……………………………... 14
D、 DNA片段化之純化及膠體電泳………………………. 15
E、 Propidium iodide (PI)定量胸腺細胞的死亡………….. 17
F、 以免疫螢光法測定淋巴細胞表面抗原……………….. 17
G、 比較小鼠對具活性的recombinant wild-type SPE B及
它的點突變不具活性的SPE B (C192S)的反應………18
結果
A群鏈球菌之小鼠感染模式………………………………….. 19
1) 皮下氣囊感染部位回抽液的菌數變化…………………… 19
2) A群鏈球菌感染後小鼠血中菌量的計數………………… 20
3) A群鏈球菌感染後小鼠器官組織的病理變化…………… 20
4) 利用親脂性染料Sudan III證實肝細胞的脂肪變性………22
5) 血清中ALT、AST及BUN的偵測………………….…… 23
6) A群鏈球菌感染後小鼠脾臟重量的變化…………….……24
7) A群鏈球菌感染對小鼠的胸腺重量及細胞的影響……….24
8) 小鼠感染A群鏈球菌後臟器中菌量的計數………………25
9) 血清及皮下氣囊抽出液中SPE B的含量…………………26
Recombinant wild-type SPE B及其mutant C192S以腹腔注
入小鼠腹腔後小鼠的致死率及組織的病理變化……………..27
總結與討論……………………………………………………..28
圖表……………………………………………………………. 37
引用文獻………………………………………………………. 63
附錄
附錄1
1-1. 菌株 ……………………………………………… 71
1-2. SPE B抗體的製備及純化 ……………………… 73
附錄2
競爭性ELISA解說圖 ………………………………….74
附錄3
3-1. 實驗試劑的準備 …………………………………. 75
3-2. 耗材 ………………………………………………. 77
3-3. 儀器 ………………………………………………..78
3-4. 試劑 ………………………………………………..80
作者簡介 82
授權書
圖目錄
Fig 1. Bacterial numbers in the air-pouch exudates after NZ131
or SW510 inoculation.
Fig 2. Bacterial numbers in the air-pouch exudates after A20 or
SW507 inoculation.
Fig 3 3. Histopathologic changes of kidney (I).
Fig 4. Histopathologic changes of kidney (II).
Fig 5. Histopathologic changes of kidney (III).
Fig 6. Histopathologic changes of liver (I).
Fig 7. Histopathologic changes of liver (II).
Fig 8. Histopathologic changes of liver (III).
Fig 9. Histopathologic changes of liver (IV).
Fig 10. Liver frozen sections staining by Sudan III from mice
72 h after PBS, NZ131 or SW510 inoculation.
Fig 11. Liver frozen sections staining by Sudan III from mice
48 h after A20 or SW507 inoculation.
Fig 12. Levels of AST and ALT in the sera from mice 48 h after
NZ131 or SW510 inoculation.
Fig 13. Levels of AST and ALT in the sera from mice 48 h after
A20 or SW507 inoculation.
Fig 14. Levels of BUN in the sera from mice 48 h after
NZ131 or SW510 inoculation.
Fig 15. Changes of spleen organ weights in mice 48 h after
PBS, A20 or SW507 inoculation.
Fig 16. Changes of thymus weights and thymocyte numbers in
mice various times after NZ131 or SW510 inoculation.
Fig 17. DNA fragmentation of thymocytes from mice 24 h after
NZ131 or SW510 inoculation.
Fig 18. Induction of thymocyte apoptosis and changes in
thymocyte subpopulations 24 h after NZ131 or SW510
inoculation.
Fig 19. Standard curve of SPE B with competition ELISA.
Fig 20. Levels of SPE B in air-pouch exudates and sera from
mice after NZ131 or SW510 inoculation.
Fig 21. Levels of SPE B in air-pouch exudates and sera from
mice after A20 or SW507 inoculation.
Fig 22. Peritoneal muscle sections from mice 30 min after
intraperitoneal injection with 300 ml (2 mg/ml) of
recombinant wild-type SPE B.
表目錄
TABLE 1. Dissemination of wild-type strains and mutants in the
blood at 48 h post-inoculation.
TABLE 2. Bacteria dissemination on various organs at 48 h post-
inoculation of NZ131 and SW510.
TABLE 3. Bacteria dissemination on various organs at 48 h post-
inoculation of A20 and SW507.
TABLE 4. Mortality in mice treated with recombinant wild-type
SPE B or its mutant (C192S).

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