出版年:

2000

研究生:

傅秀媚

研究生(英文姓名):

Hsiu-Mei Fu

論文名稱:

幽門桿菌桿狀體轉型為球形體及其特性分析

英文論文名稱:

Induction and characterization of Helicobacter pylori conversion from spiral to coccoid form

指導教授:

吳俊忠 ; 許博翔

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

s46874037

學年度:

88

語文別:

中文

論文頁數:

72

關鍵詞:

幽門桿菌 ; 球形體

英文關鍵詞:

Helicobacter pylori ; coccoid form

被引用次數:

0

[ 摘要 ]

幽門桿菌為革蘭氏陰性桿菌，已被認為是人類胃部潰瘍的主要致病原，感染此菌亦會增加胃炎及胃癌的發生率。幽門桿菌已被發現存在有兩種不同的形態，桿狀體與球形體。在體外可以藉由改變氧氣含量、pH值、培養溫度、延長培養天數或添加抗生素來誘導球形體。球形體目前在致病的角色仍不清楚，而此球形體是休眠或是趨向死亡，目前仍是意見分歧。本研究之主要目的在於建立一套快速體外誘導球形體之方法並探討它與桿狀體之表現差異。以添加或未添加抗生素kanamycin 100 mg/ml至培養液後，於一般培養箱或微需氧培養箱，進行球形體之誘導。觀察其轉型比率;並分別比較桿狀體及球形體之上清液對HeLa及AGS cells之細胞毒力能力、CLO test偵測尿素的活性、S35偵測之蛋白質新合成情況及菌液餵食小鼠，觀察細菌之附著能力及其病理變化。在光學顯微鏡下觀察，發現幽門桿菌置於一般培養箱並添加kanamycin之誘導球形體的效果最好，而球形體之表現於第三天幾近於100%。端鞭毛及細菌外觀的完整性、HeLa及AGS細胞株之細胞空泡能力與尿素的活性在球形體與桿狀體並無顯著的差異。相反的，而在球形體新合成的蛋白質則明顯高於桿狀體。在小鼠動物實驗，球形體與桿狀體幽門桿菌在BALB/c小鼠之附著能力相當。此外，在SCID小鼠病理組織觀察發現有極明顯發炎現象，且發現不論球形體或桿狀體感染，在SCID小鼠的胃中幽門桿菌附著現象皆以球形體表現。綜合以上，本研究已建立一穩定快速之幽門桿菌球形體誘導方法，此方法有助於球形體致病性與感染幽門桿菌的初期附著之深入研究。

[ 英文摘要 ]

Helicobacter pylori, a curved Gram-negative bacterium, is now recognized as the main etiologic agent of peptic ulcer diseases in human. The infection of this microorganism also increases the risk of type B gastritis and gastric cancer. In human stomach, H. pylori can exist as two different morphological forms, spiral and coccoid forms. Morphological conversion from spiral to coccoid form can be induced under several sub-optimal conditions in vitro. These conditions include aerobiosis, alkaline pH, low temperature, extended incubation or antibiotics. The coccoid form has been demonstrated to retain weak metabolic activity, double membrane system and adhesion ability. However, the exact role of coccoid form in the pathogenesis of H. pylori infection remains unclear. The aim of this study was thus to find the optimal condition for the induction of coccoid form and further characterize the role of the coccoid form of H. pylori infection. Among four methods applying to induce coccoid form in this study, the most optimal condition was described as following: the spiral form of H. pylori was inoculated to the brucella broth containing 100 μg/ml of kanamycin under aerobic condition at 37°C for three days. No spiral form of H. pylori was observed either by the light microscope or by the electron microscope (EM) after the completion of the induction. The vacuolating cytotoxin activity (VCA) on either HeLa cells or AGS cells, urease activity by CLO test, colonization of BALB/c mice with 108 CFU/ml inoculation, and morphological structures by EM were used to evaluate the pathogenic characteristics of the coccoid form. There were no significant differences between spiral and coccoid forms in the intact of cell membrane and flagella under EM observation, the urease activity, and the VCA in vitro. The de novo protein synthesis of coccoid form was even higher than those of spiral form. In in vivo study, the coccoid form of H. pylori can be successfully transformed to spiral form after colonization in BALB/c mice. The ability of bacterial colonization in the BALB/c mice were not different between coccoid form and sprial form. Moreover, although the H. pylori uniformly colonized as coccoid form in SCID mice despite of the challege with either spiral or coccoid form, the gastric mucosa showed severe inflammatory response and ulcerations in SCID mice. These findings suggest that the coccoid form may play an important role in the pathogenesis of H. pylori infection, especially the initial colonization stage for infection.