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標題: | PTEN-Akt訊息傳遞路徑在調控胃幽門螺旋桿菌引起TRAIL誘發之人類胃上皮細胞凋亡之研究 Study of PTEN-Akt Signaling Pathway in Helicobacter pylori-induced TRAIL-mediated Apoptosis in Gastric Epithelial Cells |
作者: | Szu-Ying Chen 陳思穎 |
指導教授: | 許秉寧(Ping-Ning Hsu) |
關鍵字: | 胃幽門螺旋桿菌,PTEN-Akt訊息傳遞路徑,FLIP,RAIL,細胞凋亡, helicobacter pylori,PTEN-Akt signalling pathway,FLIP,TRAIL,Apoptosis, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 胃幽門螺旋桿菌(Helicobacter pylori, HP)是一種生存於微厭氧環境下的螺旋桿菌,主要棲息在人體的胃部,且至少感染於一半以上的世界人口,是目前最常見的細菌性感染源之一。眾多研究指出,胃幽門螺旋桿菌和許多胃部疾病的發生有高度相關,如胃炎、胃潰瘍、黏膜層淋巴瘤、胃癌,並且胃幽門螺旋桿菌感染至胃部後,會誘導胃上皮細胞大量細胞凋亡,是導致胃部疾病發生原因之一。在我們實驗室先前發表的研究中發現,由感染胃幽門螺旋桿菌的病患胃部中分離出的浸潤T淋巴細胞表面會表現引起細胞凋亡之蛋白質分子-TNF-related apoptosis-inducing ligand (TRAIL)。而進一步研究發現,胃幽門螺旋桿菌會藉由降低細胞內Akt的磷酸化程度,而增加short form FLICE-inhibitory protein (FLIPS)的泛素化修飾,促使FLIPS降解而負調控胃上皮細胞FLIPS的表現,增強TRAIL receptor下游的Death-Inducing Signaling Complex (DISC)的形成,啟動Caspase8媒介之細胞凋亡。然而,胃幽門螺旋桿菌是透過何種機制調控Akt- FLIPS訊息傳遞路徑仍不清楚。
在我們的實驗結果顯示,胃幽門螺旋桿菌可能透過降低胃上皮細胞內的PTEN (Phosphatase and tensin homolog)磷酸化程度,增強PTEN之去磷酸酶的活性,而降低Akt的磷酸化程度,因而調控FLIPS的表現。利用質體轉染使PTEN在胃上皮細胞過度表現後,可以發現Akt磷酸化程度及FLIPS表現量下降,並且隨著TRAIL刺激濃度的升高,細胞凋亡程度有增加的趨勢;反之利用siRNA抑制PTEN表現則可以阻斷胃幽門螺旋桿菌所引起的Akt去磷酸化、FLIPS降解現象及減緩細胞凋亡。此外,使用PTEN上游分子Casein Kinase 2(CK2)之抑制劑TBB,以抑制PTEN磷酸化,可以發現類似胃幽門螺旋桿菌感染之情況,在TRAIL刺激下細胞凋亡程度顯著提升。上述結果指出胃幽門螺旋桿菌可能會藉由干擾胃上皮細胞內PTEN-Akt- FLIPS之訊息傳遞路徑,而影響細胞對於TRAIL之敏感性。 先前實驗室的初步結果暗指Akt可能是透過AIP4調控FLIPS。我們利用CRISPR技術將AIP4基因自AGS細胞剔除後,受胃幽門螺旋桿菌感染之AGS細胞在TRAIL刺激下,細胞凋亡程度減緩,顯示AIP4亦參與在胃幽門螺旋桿菌引起TRAIL誘發之人類胃上皮細胞凋亡。 以上發現指出胃幽門螺旋桿菌的致病機制,並提供將來治療胃幽門螺旋桿菌感染相關疾病治療之重要標的。 Helicobacter pylori is a Gram-negative, microaerophilic spiral-shape bacterium and it is a common pathogen inhabiting in the gastrointestinal tract and is infected in at least half of the world’s human population. Several studies have reported that H. pylori-induced apoptosis in gastric epithelial cells is highly associated with the pathogenesis of various gastrointestinal diseases such as gastritis, peptic ulcers, gastric MALT lymphomas, and gastric cancer. Our lab previously found that infiltrating T cells from gastric mucosa of H. pylori-infected patients express TNF-related apoptosis-inducing ligand (TRAIL), which is a protein that can induce the apoptosis in several cell lines. Furthermore, our preliminary data showed that H. pylori enhances the degradation of short form FLICE-inhibitory protein (FLIPS), which inhibits TRAIL-induced apoptosis, by increasing the ubiquitination of FLIPS through decreasing the phosphorylation of Akt in human gastric epithelial cells. FLIPS downregulation enhances the assembly of Death-Inducing Signaling Complex (DISC) which activates caspase-8 to initiate apoptosis-inducing caspase cascade. However, the mechanisms by which H. pylori regulates Akt-FLIPS pathway remain unclear. Here we showed that H. pylori downregulates PTEN phosphorylation level, which activates the phosphatase activity of PTEN, to dephosphorylate Akt and consequently decreases FLIPS expression level. Overexpression of PTEN resulted in Akt dephosphorylation, FLIPS degradation and the increasing of TRAIL-mediated apoptosis in a dose-dependent manner. Moreover, the H. pylori induced-apoptosis signaling and H. pylori-induced TRAIL-mediated apoptosis were inhibited by knockdown of endogenous PTEN. Furthermore, we demonstrated that PTEN activation with treatment of Casein Kinase 2 inhibitor enhanced TRAIL-mediated apoptosis. Our results indicated that H. pylori regulates the sensitivity to TRAIL-induced apoptosis through PTEN-Akt- FLIPS pathway in gastric epithelial cells. Previous preliminary data from our lab showed AIP4 may involve in Akt- FLIPS pathway. To further validate the role of AIP4, we generated AIP4 knockout AGS single cell clones by CRISPR/Cas9. The H. pylori-induced TRAIL-mediated apoptosis was inhibited in AIP4 KO cells, but not wild type AGS cells, indicating that AIP4 also participates in H. pylori-induced TRAIL-mediated apoptosis. These findings explore the mechanism of H. pylori pathogenesis and provide potential therapeutic targets for treatment of H. pylori-related disease. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68368 |
DOI: | 10.6342/NTU201703511 |
全文授權: | 有償授權 |
顯示於系所單位: | 免疫學研究所 |
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