出版年:

研究生:

林詩婷

研究生(英文姓名):

Shih-Ting Lin

論文名稱:

I.FldA及免疫球蛋白重鏈基因於胃黏膜相關性淋巴組織淋巴瘤中所扮演之角色 II.探討FlgK於幽門桿菌感染中之作用

英文論文名稱:

I.The roles of FldA protein and heavy chain of immunoglobulin gene in the gastric MALToma. II. The effect of FlgK in Helicobacter pylori infection

指導教授:

許博翔 ; 吳俊忠

指導教授(英文姓名):

Bor-Shyang Sheu ; Jiunn-Jong Wu

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物及免疫學研究所

學年度:

90

語文別:

中文

論文頁數:

128

關鍵詞:

鞭毛 ; 免疫球蛋白重鏈基因 ; 黏膜相關性淋巴組織淋巴瘤 ; 幽門桿菌

英文關鍵詞:

PCR clonality ; Helicobacter pylori ;
mucosa-associated lymphoid tissue lymphoma (MALT ;
flagellum

被引用次數:

0

[ 摘要 ]

幽門桿菌是一株革蘭氏陰性且必須生長於微需氧環境的一隻細菌，屬於人類胃腸部之病原菌。目前知道幽門桿菌與一些胃腸疾病之發生有關，如胃潰瘍、十二指腸潰瘍、胃癌以及黏膜相關性淋巴組織淋巴瘤。先前有報導指出一個大小19 kDa的幽門桿菌蛋白FldA與黏膜相關性淋巴組織淋巴瘤之形成有關連，對於此蛋白是否真的與黏膜相關性淋巴組織淋巴瘤形成有關，或能否應用於預測黏膜相關性淋巴組織淋巴瘤之復原程度仍然是個未知數。因此在論文的第一部份以純化之FldA蛋白利用西方墨點法進行病人血清分析，此外利用免疫球蛋白重鏈基因的PCR分析淋巴瘤中B淋巴球的clonality。結果顯示抗FldA抗體在黏膜相關性淋巴組織淋巴瘤患者中出現之機率較對照組高(41.2% vs 8.7%)，而即使在黏膜相關性淋巴組織淋巴瘤完全消退之病人身上，其抗FldA抗體會持續存在至少有12個月，由這個數據顯示抗FldA抗體或許可以當成一個血清標誌，但並不是一個主要決定淋巴瘤消退之因子。經由淋巴瘤組織所萃取之染色體，結果顯示約有八成的病人在治療前呈現monoclonality，既使經病理學判定為完全恢復之病人中仍有三分之一為monoclonality。此結果顯示PCR clonality也不適合用於預測黏膜相關性淋巴組織淋巴瘤之恢復情況。
幽門桿菌的鞭毛對於細菌建立菌落叢扮演一個重要之角色，目前知道在鞭毛之合成需要四十多個基因之參與，FlgK為一構成鞭毛之蛋白，經序列分析知道其與沙門桿菌的Hook-associated protein 1(HAP1)具相似性。在實驗室先前的研究發現在flgK基因被中斷後，幽門桿菌缺乏鞭毛之形成且喪失運動能力，同時在小鼠的感染模式中也發現flgK變異株在胃黏膜層所造成之高密度菌體附著量比野生株少。目前FlgK蛋白在幽門桿菌中所扮演的角色仍不清楚，所以在論文的第二部份為構築FlgK蛋白之表現質體，並且將此重組FlgK蛋白純化出來做進一步之分析。藉由將flgK基因植入pET-30b表現質體而成功地得到重組FlgK蛋白，再利用鎳離子親和管柱將重組FlgK蛋白純化出，經由西方墨點法及N端胺基酸序列分析確認其序列之正確性。抗重組FlgK蛋白之多株抗體也製備成功，利用此多株抗體的存在下可於體外抑制幽門桿菌之運動性。在小鼠研究上設計三組實驗分別為給予小鼠野生株及變異株，或給予重組FlgK蛋白免疫過小鼠野生株。結果發現在免疫過小鼠的胃中，幽門桿菌的菌落密度在一週時比四週時高。以ELISA分析各組小鼠之血清，發現在免疫過小鼠中其抗重組FlgK蛋白抗體量較餵與野生株組高，而比較免疫過小鼠在一週與四週之差異，則看到在一週時抗體量較高。分析在免疫過小鼠所引起之專一性淋巴球對於重組FlgK蛋白之反應，結果可以看到免疫小鼠在第一週時的增生現象低於野生株組，但在四週時則明顯高於野生株組。由抗體強度、淋巴球及菌落密度三者相互結合比較，顯示以重組FlgK蛋白免疫小鼠具有降低幽門桿菌感染之菌落密度，且對重組FlgK蛋白具專一性反應之淋巴球可能扮演主要之角色。
總和以上所述，在本論文中看到抗FldA抗體或許可以當成一個血清標誌，但並不是一個主要決定淋巴瘤消退之因子，且PCR clonality不適合用於預測黏膜相關性淋巴組織淋巴瘤之恢復情況；另一方面以rFlgK免疫過之小鼠可降低幽門桿菌感染之菌落密度。

[ 英文摘要 ]

Helicobacter pylori is a gram-negative, microaerophilic bacterium and human gastrointestinal pathogen. The bacterium causes gastric ulcers, duodenal ulcers, gastric cancer, and mucosa-associated lymphoid tissue lymphoma (MALToma). Previous reported that the development of gastric MALToma is associated with a 19 kDa protein (FldA) of H. pylori. Whether this protein can predict or correlate with regression of the MALToma remains unclear. The first part of my project was to purify the FldA protein and analyze patient’s serum by Western blotting. In addition, I also detected the clonality of immunoglobin heavy chain from MALToma tissues with PCR. The prevalence rate of anti-FldA antibodies was higher in MALToma patients’s sera than in the control sera (41.2 vs 8.7%). The anti-FldA antibodies persisted until the end of the 12 months even after the complete regression of MALToma. These data suggest that anti-FldA antibodies might serve as a serological marker but not the determinant of tumor regression. Analysis of the DNA extracted from serial MALToma’s tissues revealed that 80% of MALToma patients before treatment exhibited PCR monoclonality. Persisted monoclonality was found in nearly 1/3 of patients despite of the histological regression suggesting that the clonality could not be used to predict the regression of MALToma patients.
The flagellum of H. pylori has been shown to play an important role in colonization. There are more than forty genes involved in the biosynthesis of flagella. The FlgK protein, a component of flagella, is a homologue of hook-associated protein 1 (HAP1) of Salmonella. In our laboratory, the flgK isogenic mutant was constructed. Neither flagella nor motility was in the mutant. In the murine model, the isogenic strain also showed less colonization density than that of wild-type strain in the mucus layer of stomach. At present, the role of FlgK in H. pylori is still unknown. The second part of my project was to construct the FlgK expression vector and purify this protein for further studies. The flgK gene was inserted into pET-30b expression vector and the recombinant FlgK (rFlgK) protein was expressed successfully. After purified by Ni2+-affinity column, the rFlgK protein was confirmed by Western blot and N-terminal amino acid sequence analysis. The polyclonal antibody against rFlgK was also produced. The motility of H. pylori was inhibited in the medium mixed with rFlgK polyclonal antibodies. In murine model, three groups of experiments were designed including mice were inoculated with wild-type and mutant-type of strain and rFlgK immunized mice were inoculated with wild-type strain. It was found that the colonization density of H. pylori in 1 week was higher than in 4 weeks in rFlgK immunized mice. The anti-rFlgK antibody titers in immunized mice were higher than that of unimmunized mice. Compare the titers between 1 and 4 weeks in immunized mice, the antibody titers were higher in 1 week. To analyze the specific lymphocyte response triggered by rFlgK in immunized mice. There were significantly lower proliferation responses in 1 week than unimmunized mice. Whereas, the proliferation responses were significantly higher in 4 weeks. These data showed that rFlgK immunization could reduce the colonization density and specific lymphocyte response may play the major role.
In conclusions, anti-FldA antibodies might can as a serological marker but not the determinant of tumor regression. In addition, PCR clonality cannot be used to predict the regression of MALToma. The H. pylori colonization density can be reduced in rFlgK immunized mice.