臺灣博碩士論文加值系統

幽門桿菌為一隻革蘭氏陰性桿菌，屬於微需氧且具有螺旋狀外型，能夠群居在人類胃壁黏膜處。一些與附著相關的基因被認為與該細菌的致病能力有關。近來發現一個被命名為 sialic acid-binding adhesin (SabA) 的附著分子會和細胞表面的 sialyl di-Lewis x 抗原 (sdiLex) 結合，並且此結合作用發生在幽門桿菌先以另一被研究較多的附著分子 BabA 與Lewis b 抗原結合之後。目前 SabA 蛋白在台灣菌株的情形，以及當宿主較少或不表現 Lewis b 抗原時，SabA 的角色以及與臨床病徵的關聯均不清楚。本實驗以148株臨床菌株進行 PCR 偵測 sabA 基因的存在，結合兩對引子的結果得到80.4%的陽性率 (119/148)。119株陽性菌株中有95株的 PCR 產物能成功定序，再依有無明顯之 CT 雙核苷酸重複序列可歸為2種類型，其中37株不含 CT 重複序列而歸為第二型者，有36株依照可完全轉譯和可能具有 signal sequence 推測其會表現蛋白。再加上18株具有 CT 重複序列且表現 SabA 的第一型菌株，推測在台灣菌株中 SabA 蛋白的盛行率為43.5% (54/124)。流行病學調查中發現，當 SabA 蛋白與宿主 Lewis x 抗原均表現時，宿主 Lewis b 抗原的表現較弱;若兩者共同表現則與宿主胃菌落量的增加有關。先前研究指出 hopZ 變異株在體外實驗中附著到胃細胞株的能力有顯著下降，並且對 C57BL/6 小鼠胃的群居量也較少，但目前並不清楚 HopZ 蛋白在臨床病人胃部感染時是否也扮演著附著的角色。和部分 sabA 基因相同，hopZ 基因的表現由位於該基因5'區域之不同數目的 CT重複透過 slipped-strand mispairing 機制所調控，故在臨床菌株的 hopZ 基因中可發現有不同的表現狀態。本實驗共收集148株臨床菌株進行 PCR 以偵測 hopZ 基因，結果陽性菌株佔83.1% (123/148)。123株陽性菌株中有92株的 PCR 產物成功被定序，其中33株依 CT 重複數目配合可完整轉譯之觀察預測為可以表現 HopZ 蛋白，另有兩株CT 重複數目原屬 “off” 者的 DNA 序列亦可被完整轉譯，因此推測 HopZ 蛋白在台灣菌株的盛行率為29.9% (35/117)。然而感染 HopZ表現菌株與宿主病理變化除 Lewis x 抗原表現量略高外並無其他明顯關聯。本實驗亦用基因置換方式成功建立3株 hopZ 變異株，並觀察其附著能力的改變。結果發現野生株與變異株附著到 AGS 細胞的程度並無不同，即使在BabA 的功能被抑制後兩者的附著程度亦無差異。根據以上結果，本研究推論 SabA 在宿主缺乏或少量表現Lewis b 抗原時，能幫助細菌的附著進而群居在宿主胃中;但 HopZ 可能在幽門桿菌附著到胃上皮細胞的過程並非扮演重要角色。

Helicobacter pylori is a Gram-negative, microaerophilic spiral bacterium which colonizes the mucosa of human stomach. Genes involved in adhesion is thought to be essential for virulence. One adhesin, named sialic acid-binding adhesin (SabA), was recently found that it binds to sialyl di-Lewis x antigen (sdiLex) on host cell surface. This binding occurs after H. pylori using the well-demonstrated BabA adhesin for binding to Lewis b antigen. The prevalence of SabA protein in Taiwanese strains, and the association of SabA with the clinical outcomes when host Lewis b expression is weak or absent are not clear. A total of 148 domestic strains were enrolled for detecting sabA gene by PCR. Combining the results of two primer pairs, the probable genopositive rate of sabA gene was 80.4% (119/148). The PCR products of sabA from 95 of 119 genopositve strains were successfully sequenced. These sabA sequences were classified into 2 types according to the existence of obvious CT dinucleotide repeat tract in the signal sequences of them. Among 37 of type II strains without CT repeats, 36 were proposed to be able to express SabA protein by the evidences of the in-frame translation and the existence of putative signal sequence. Including the 18 SabA-expressing type I strains with CT repeat tract, the prevalence rate of putative SabA protein in Taiwanese strains was 43.5% (54/124). Reduced expression of host Lewis b expression was found when SabA and host Lewis x antigen were simultaneously expressed. Furthermore, simultaneous expression of SabA and host Lewis x was associated with enhanced bacterial density in host stomach. Previous reports described that the isogenic hopZ mutant showed significantly reduced binding to gastric cell line in vitro and lower the colonization density to stomach of C57BL/6 mice. However, the actual role of HopZ protein for colonization is not clear in clinical patients. The phenotypic variation of the hopZ gene, as same as a portion of sabA genes, is regulated by different CT repeats within 5’ region of the gene through slipped-strand mispairing mechanism. Therefore, “on” or “off” of hopZ gene expression can be disclosed in the different clinical isolates. A total of 148 H. pylori isolates were also detected for hopZ gene by PCR. The genopositive rate of hopZ gene was 83.1% (123/148). The PCR products from 92 of 123 genopositve strains were successfully sequenced for hopZ sequence. Among these 92 strains, 33 strains were disclosed with HopZ protein expression predicted by the number of repeats matching the in-frame translation. DNA sequences of two strains with the repeat number originally for “off” status could also be translated completely. Therefore, the probable prevalence rate of HopZ protein in Taiwanese strains was 29.9% (35/117). However, there was not any correlation between the infection of HopZ-expressing strains and the pathological changes except Lewis x expression was slightly enhanced in host stomach. Three isogenic hopZ mutants were successfully constructed by gene replacement and assayed for their adhesion ability. There was no difference between wild-type strains and mutants in adherence to AGS cell line even if the function of BabA was blocked. Based on the results above, SabA may help bacterial adherence for colonization in host when host Lewis b expression is weak or absent, but HopZ may not have an important role in adhesion of H. pylori to gastric epithelium.

中文摘要……………………………………………… i
英文摘要……………………………………………… iii
誌謝…………………………………………………… vi
目錄…………………………………………………… vii
表目錄…………………………………………………… x
圖目錄………………………………………………… xii
符號與縮寫……………………………………………xiii
緒論…………………………………………………… 1
材料與方法…………………………………………… 20
一、細菌、細胞株與質體………………………… 20
二、儀器與藥品…………………………………… 20
三、細菌之鑑定、培養與保存…………………… 20
四、細胞的培養和保存…………………………… 21
五、宿主之病理及組織學診斷…………………… 22
六、細菌DNA之抽取………………………………… 24
七、聚合酶連鎖反應 (PCR)……………………… 25
八、核酸定序 (DNA sequencing) 分析………… 26
九、統計及電腦分析……………………………… 27
十、hopZ 變異株之構築…………………………… 27
十一、南方墨漬雜交法 (Southern blotting
hybridization)…………………………… 30
十二、附著能力試驗 (Adhesion assay)………… 32
結果…………………………………………………… 35
部分一: sabA 基因………………………………… 35
一、sabA 基因在台灣幽門桿菌臨床菌株之流行
率…………………………………………… 35
二、sabA 基因5'端序列之變異………………… 36
三、Type II sabA 序列之分析………………… 37
四、SabA 蛋白在臨床菌株之表現情形………… 39
五、sabA 基因之表現與病患胃組織病變之關聯 41
六、SabA 分子與缺乏或 Lewis b 表現弱之病
患胃組織病變之關聯……………………… 42
部分二: hopZ 基因………………………………… 44
一、hopZ 基因在台灣幽門桿菌臨床菌株之流行
率…………………………………………… 44
二、hopZ 基因CT雙核苷酸重複序列之分佈與
HopZ蛋白表現情形………………………… 44
三、hopZ 基因之表現與宿主胃組織病理變化
之關聯性…………………………………… 46
四、hopZ 基因變異株之構築…………………… 47
五、使用南方墨漬法確認 hopZ 基因變異株的建
立…………………………………………… 49
六、hopZ 基因之變異對附著能力的影響……… 50
討論…………………………………………………… 52
參考文獻……………………………………………… 59
圖表…………………………………………………… 73
附錄…………………………………………………… 116
自述…………………………………………………… 131

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