胃幽門螺旋桿菌在調控細胞活化與凋亡之研究

Abstract

胃幽門螺旋桿菌的感染與慢性胃炎和消化性潰瘍甚至是胃癌和胃粘膜相關淋巴組織淋巴瘤的疾病發生有關。胃幽門螺旋桿菌引發胃部疾病的致病性機轉主要可分成兩個部份來探討：一部分是胃幽門螺旋桿菌本身所具有的毒性因子所造成的毒殺作用，另一部份則是引發宿主產生的發炎反應所造成的影響。胃幽門螺旋桿菌造成疾病發生的角色已被證實，但引發致病性的相關機轉仍不清楚。 首先，為了探討胃幽門螺旋桿菌對於腫瘤發生的相關機轉，我們探討胃幽門螺旋桿菌CagA蛋白對於粘膜相關淋巴組織淋巴瘤發生機轉所扮演的角色。先前的研究報告指出胃幽門螺旋桿菌可以藉由依附胃上皮細胞將CagA蛋白送入細胞中，被送入細胞內的CagA蛋白可以調節細胞內的訊息傳導路徑並且引發致病性，因此我們認為在胃幽門螺旋桿菌感染的過程中，可能藉由與B細胞的直接作用來造成粘膜相關淋巴組織淋巴瘤。我們證實胃幽門螺旋桿菌可以將CagA蛋白送入人類B細胞中，並且在細胞內進行酪氨酸的磷酸化作用後，與細胞內的SHP-2進行結合，另外，CagA蛋白還會活化細胞內的ERK與p38 MAP kinase，並且造成細胞內存活相關因子Bcl-2和Bcl-XL的表現量增加，進一步防止細胞死亡。本研究顯示胃幽門螺旋桿菌可以直接將CagA致癌蛋白送入人類的B細胞中，造成粘膜相關淋巴組織淋巴瘤及病的發生。 第二，為了探討宿主因子在胃幽門螺旋桿菌引發疾病的過程中所扮演的角色，於是我們研究胃幽門螺旋桿菌所引起細胞凋亡的現象之中TRAIL分子扮演的角色。在我們先前的研究指出，胃幽門螺旋桿菌可以改變胃上皮細胞對於TRAIL的感受性改變，使細胞進行細胞凋亡，我們進一步研究指出胃幽門螺旋桿菌可引發TRAIL所造成細胞內caspase-8、casapse-3以及mitochondria的活化，因而誘發胃上皮細胞的細胞凋亡作用，同時也發現，胃幽門螺旋桿菌可加強TRAIL所引發death inducing signaling complex (DISC) 的聚集，因此引發足夠多的caspase-8活化，因而打破原本的抗性，造成細胞的敏感性增加，此過程是經由胃幽門螺旋桿菌誘發c-FLIPS的表現量降低所造成，此外，在細胞內過度表現c-FLIPS可以阻斷TRAIL所引起的細胞凋亡，進一步單獨利用c-FLIPS siRNA就可以改變細胞對於TRAIL的感受性，證實其重要性。因此，胃幽門螺旋桿菌可以經由降低細胞內c-FLIPS的表現，因而加強細胞內DISC的聚集，藉此活化較多caspase-8，因而調控胃上皮細胞對於TRAIL的感受性。 總結，致病菌因子與宿主因子在胃幽門螺旋桿菌所導致的疾病發展過程中扮演重要的角色，對於胃幽門螺旋桿菌引發疾病的過程中是很重要的致病機轉。

Infection with the gastric pathogen H. pylori infection has been associated with the development of chronic gastritis and peptic ulcer diseases, moreover, gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori infection leads to pathogenesis of gastric diseases mainly by two different mechanisms : direct toxicity of H. pylori virulent factors and induction of inflammatory responses. The role of H. pylori in association with disease development has been demonstrated, however, the mechanisms underlying pathogenesis are still unclear. First, in order to investigate the mechanisms underlying H. pylori induces the tumourgenesis, we examine the role of H. pylori CagA in the development of MALT lymphoma. Previous studies reveal that H. pylori attaches to the epithelial cells, and translocates CagA proteins into cells. Intracellular CagA deregulates intracellular sighaling pathway and causes the pathogenic effects. This in turn raised the possibility that H. pylori is associated with the development of MALT lymphomas during persistent infection by direct interaction with B lymphocytes. We demonstrated that H. pylori directly translocates CagA into human B cell line and primary B cells. Furthermore, injected CagA undergoes tyrosine phosphorylation and interacts with SHP-2. Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase were activated upon CagA translocation. The cell survival factors, such as Bcl-2 and Bcl-XL were upregulated through the translocated CagA, moreover, prevents apoptosis. These results provide evidence that CagA is directly delivered into B cells by H. pylori and acts as a bacterium-derived oncoprotein in human B cells to cause the development of MALT lymphoma. Second, to study the role of host factors in regulating H. pylori-induced gastric diseases, we investigated the role of TNF related-apoptosis inducing ligand (TRAIL) in the induction of apoptosis during H. pylori infection. In our previous study, we provided the evidences that H. pylori can sensitize human gastric epithelial cells to TRAIL-mediated apoptosis. Further, we demonstrated that H. pylori could sensitize gastric epithelial cells to TRAIL, resulting in caspase-8, caspase-3 and mitochondrial pathway activation and induce apoptosis. H. pylori could enhance the death inducing signaling complex (DISC) assembly and induce sufficient amount of caspase-8 activation to break apoptosis resistance, inducing the TRAIL sensitivity via downregulation of the c-FLIPS in gastric epithelial cells. Overexpression of c-FLIPS could block apoptosis induced by TRAIL in the presence of H. pylori. Moreover, silencing c-FLIPS expression by siRNA increased TRAIL-induced apoptosis in human gastric epithelial cells. In summary, our data suggest that H. pylori could downregulate the expression level of c-FLIPS, resulting in enhanced DISC assembly and caspase-8 activation to modulate TRAIL-mediated apoptosis in gastric epithelial cells. Taken together, pathogen and host factors are critical in the development of H. pylori-associated diseases, the interplay between H. pylori and immune cells as well as gastric epithelial cells play important roles in the pathogenesis of H. pylori-mediated gastric disease.