指導教授:許秉寧臺灣大學:免疫學研究所唐承希Tang, Chen-HsiChen-HsiTang2014-11-262018-07-092014-11-262018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/262024胃幽門螺旋桿菌(Helicobacter pylori, HP)為常見的人類病原菌,目前已知感染全球約50%的人口,並增加感染者罹患十二指腸潰瘍、胃潰瘍、胃癌及黏膜相關淋巴癌的機率。研究指出,胃幽門螺旋桿菌感染引起的胃部細胞凋亡是引起胃潰瘍的主因之一。在我們的研究顯示,從HP病患之胃部組織分離出的T淋巴球,其細胞表面表現TRAIL蛋白質,TRAIL為能夠引起細胞凋亡的蛋白質之一。我們進一步發現,胃幽門螺旋桿菌調節TRAIL引起的人類胃上皮細胞凋亡訊息,HP引發TRAIL的敏感性主要是依靠負調控short form of FLICE-inhibitory protein (FLIPs)而增強TRAIL死亡誘導訊息複合物(Death-Inducing Signaling Complex, DISC)的形成,並導致caspase-8及其下游路徑的活化而引起細胞凋亡。 在此論文中,我們進一步研究影響宿主細胞凋亡訊息的HP因子,我們使用CAG pathogenic island(CAG PAI)基因剔除的菌株來篩選關鍵的因子,結果顯示HP依靠第四型分泌系統而引起細胞凋亡訊息。我們發現造成第四型分泌系統缺陷的基因突變菌株無法引起細胞對於TRAIL的敏感性。此外,這些突變菌株失去負調控細胞內FLIPs表現的能力,因此在TRAIL刺激時無法引起人類胃上皮細胞中caspase-8活化。相對地,CagA缺陷不影響HP負調控FLIPs及引起細胞對TRAIL的感受性。這些結果顯示HP的第四型分泌系統在此現象中的重要性,並且非由CagA所引起。 此外,在宿主細胞方面,我們也探討可能受到HP干擾並影響FLIPs 表現的訊息路徑。我們的結果顯示HP抑制Akt (protein kinase B)活化,並且Akt的活性與FLIPs表現具有相關性。若抑制Akt的活性或增加Akt的表現,能使FLIPs表現量相對地減少及增加。因此我們進一步在胃上皮細胞中過度表現Akt,研究HP是否透過干擾Akt訊息途徑而影響細胞對TRAIL感受性。結果發現細胞表現過量Akt時,能減緩HP引起的細胞凋亡程度,因此這些結果顯示HP可透過干擾Akt訊息途徑而改變人類胃上皮細胞FLIPs表現,因此提升細胞對TRAIL感受性並造成細胞凋亡。此研究幫助我們了解HP引起細胞凋亡的機制,並提供關鍵的因子作為治療HP相關疾病的依據。Helicobacter pylori (HP), a common human pathogen which infects about 50% of the world’s population, is associated with duodenal ulcer, peptic ulcer diseases, gastric cancer and Mucosa-associated lymphoid tissue lymphoma (MALToma). Gastric cells apoptosis induced by HP were reported to be involved in the pathogenesis of peptic ulcer. Our previous studies have demonstrated that T lymphocytes from gastric tissue of HP patients expressed TRAIL, which could induce apoptosis. Furthermore, HP regulates TRAIL apoptosis signaling in human gastric epithelial cells by down-regulation of cellular short form of FLICE-inhibitory protein (FLIPs), which leads to enhanced assembly of the TRAIL death-inducing signaling complex (DISC) and caspase-8 activation and eventually apoptosis. In this thesis, we further study the HP factors which regulated the apoptosis signaling in host cells, HP mutants with gene deficiency in the cytotoxin-associated gene pathogenicity island (CagPAI) were screened to identify the key factors in inducing apoptosis. Our data revealed that the induction of apoptosis signaling is dependent on the type IV secretory system of HP. We found that mutant strains with deficient type IV secretion system lost the ability to induce TRAIL sensitivity. Furthermore, these mutant strains were unable to down-regulate FLIPs and caspase-8 activation upon TRAIL treatment in human gastric epithelial cells. In contrast, CagA mutation showed no effect on HP-induced FLIPs down-regulation and TRAIL sensitivity. These results indicated that type IV secretion system of HP, but not CagA, is critical. In addition, in host cells aspect, we also explore the signaling pathway that HP may disturb to down-regulate FLIPs expression. Our results indicated that HP suppressed Akt (protein kinase B) activation, and there is a correlation between Akt activity and FLIPs expression in human gastric epithelial cell line. FLIPs expression was down- or up-regulated respectively by inhibiting Akt activity or over-expressing Akt. We further investigate whether HP disturbed Akt signaling pathway to induce TRAIL sensitivity by using Akt-overexpressing gastric epithelial cells. Over-expression of Akt mitigated HP-induced apoptosis, so these results indicated that HP modulated FLIPs expression to enhance TRAIL-mediated apoptosis partially by disturbing Akt signaling pathway. This study helps us to understand the mechanisms in inducing apoptosis by HP, and thereby to identify key factors which may help to design treatment of HP-associated diseases.致謝………………………………………………………………..……..ii 摘要……………………………………………………………………...iii Abstract………………………………………………………………….v 研究背景…………………………………………………………………1 1. 胃幽門螺旋桿菌簡介………………………………………………..1 2. CagPAI與第四型分泌系統………………………………………….1 3. 胃幽門螺旋桿菌感染引起細胞凋亡………………………………….2 4. TRAIL與細胞凋亡…………………………………………………3 5. 胃幽門螺旋桿菌促進TRAIL死亡誘導訊息複合物(DISC)之形成而引起細胞凋 亡…………………………………………………………...4 6. Akt調控FLIP並影響細胞凋亡啟動………………………………….4 研究動機與目的…………………………………………………………6 實驗材料與方法…………………………………………………………7 1. 實驗材料…………………………………………………………...7 2. 實驗方法………………………………………………………….13 實驗結果………………………………………………………………..16 1. 胃幽門螺旋桿菌能增進胃上皮細胞對於TRAIL之感受性,然而當胃幽門螺旋桿菌 致病因子CagPAI及CagE基因有缺陷時,即失去此特性……16 2. 胃幽門螺旋桿菌致病因子CagPAI及CagE在負調控胃上皮細胞FLIPs表現量並 造成Caspase-8活化的現象中扮演重要的角色………………..16 3. 胃幽門螺旋桿菌與空腸曲桿菌皆具有第四型分泌系統,然而空腸曲桿菌並無法有 效增加胃上皮細胞對於TRAIL之感受…………..…………..17 4. 在胃上皮細胞株AGS中,Akt的活性調控FLIPs的表現,並且胃幽門螺旋桿菌降 低胃上皮細胞中Akt活性…………………………………17 5. 在人類胃上皮細胞株AGS中,過度表現Akt可抑制胃幽門螺旋桿菌引起之TRAIL 敏感性及伴隨的細胞凋亡現象………………………..….18 結果討論 1. 胃幽門螺旋桿菌致病因子CagPAI及CagE能增進胃上皮細胞對於TRAIL之感受 性而造成細胞凋亡………………………………………….19 2. 第四型分泌系統在胃幽門螺旋桿菌造成的TRAIL敏感性現象中可能扮演的角 色…………………………………………………………….20 3. 胃幽門螺旋桿菌抑制Akt活性而負調控FILPs表現,並影響胃上皮細胞對TRAIL 之感受性………………………………………………..21 4. Akt及其它調控FLIP表現之細胞訊息途徑………………………….22 5. 結論………………………………………………………………22 參考文獻………………………………………………………………..23 圖表說明………………………………………………………………..291249049 bytesapplication/pdf論文使用權限:同意無償授權胃幽門螺旋桿菌細胞凋亡[SDGs]SDG3研究誘發TRAIL媒介的胃上皮細胞凋亡之胃幽門螺旋桿菌因子Study of Helicobacter pylori factors in H. pylori-induced sensitivity to TRAIL-mediated apoptosis in human gastric epithelial cellsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262024/1/ntu-103-R01449006-1.pdf