臺灣博碩士論文加值系統

胃幽門螺旋桿菌(Helicobacter pylori, H. pylori)為一種微厭氧的革蘭氏陰性菌，由Barry Marshall和Robin Warren從胃炎和消化性潰瘍病患身上分離出來。根據統計，全球50%以上的人口曾受到其感染，而當中超過80%的人口是沒有任何病徵的。研究指出，胃幽門螺旋桿菌不僅與胃炎和消化性潰瘍相關，更是導致胃癌的重要危險因子之一。實驗室的先前研究指出，胃幽門螺旋桿菌透過降解short form cellular-FLICE inhibitory protein (FLIPs)的方式，促使death-inducing signaling complex (DISC)的組成，進而導致caspase 8的活化，使得胃上皮細胞更容易受到tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)媒介的細胞凋亡。同時，實驗室的初步結果亦指出，透過負調控Akt的訊息傳遞路徑，胃幽門螺旋桿菌敏化TRAIL媒介的細胞凋亡。先前研究亦指出，由於內質網(endoplasmic reticulum, ER)內錯誤摺疊蛋白累積所產生的內質網壓力(ER stress)，不僅與胃幽門螺旋桿菌的致病機制有相當的關連性；另外，研究也已經指出內質網壓力與細胞凋亡間有交互作用；在胃幽門螺旋桿菌感染間，胃幽門螺旋桿菌會引起內質網壓力並且導致細胞死亡。先前研究也指出內質網壓力的發生，會透過GSK-3β介導的Rictor蛋白磷酸化，抑制mTORC2/Akt的訊息傳遞路徑。在此論文中，我們探討胃幽門螺旋桿菌對於內質網恆定的擾動是如何敏化TRAIL媒介胃上皮細胞之凋亡。首先在胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡中，我們觀察到GRP78的顯著下調以及CHOP的表現提升。另外，我們觀察到在與胃幽門桿菌共培養九小時後，GRP78和FLIPs的表現量同時顯著下調。由於GRP78的降解，反映內質網內的恆定作用受到擾動，而引起下游未摺疊蛋白質(Unfolded protein response, UPR)訊息傳遞路徑的活化。我們進而探討抑制內質網壓力對於胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡的影響，使用thapsigargin誘導胃上皮細胞產生內質網壓力。實驗結果觀察到誘導內質網壓力的發生使得FLIPs的表現量下調，並且促使胃上皮細胞發生TRAIL介導的細胞凋亡。於是，我們進一步探討下游未摺疊蛋白的訊息傳遞路徑是否參與胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡的調控，發現eIF2α的磷酸化顯著上調，並且在利用salubrinal間接抑制eIF2α的活性後，減緩胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡。透過siRNA抑制eIF2α基因表現，我們發現胃幽門螺旋桿菌造成FLIPs降解和capase8的剪切能夠被恢復，並且減緩其敏化的TRAIL介導胃上皮細胞凋亡。最後，透過siRNA抑制Akt基因表現，我們發現eIF2α的磷酸化增強以及造成FLIP的下調。綜合以上實驗結果，我們發現胃幽門螺旋桿菌透過去磷酸化Akt調控eIF2α的磷酸化，誘導FLIPs的降解，從而敏化TRAIL媒介胃上皮細胞之凋亡。這些結果有助於理解胃幽門螺旋桿菌造成細胞凋亡的機制，提供未來臨床治療的標的及方向。

Helicobacter pylori (H. pylori), a Gram-negative microaerophilic bacterium identified by Barry Marshall and Robin Warren in patients with gastric and peptic ulceration, infects about half of world population. H. pylori infection is not only associated with duodenal and peptic ulcer diseases but also is considered as the main cause of gastric cancer. However, over 80% of the individuals infected with H. pylori are asymptomatic. Previously, our lab reported that the infiltrating T lymphocytes on gastric mucosa during H. pylori infection express TNF-necrosis factor-related apoptosis inducing ligand (TRAIL), which belongs to the TNF superfamily and shares strong homology with FasL that causes apoptosis in a variety of transformed cells. Our lab further demonstrated that H. pylori sensitizes TRAIL-mediated apoptosis in human gastric epithelial cells through down-regulating the short form cellular-FLICE inhibitory protein (FLIPs) and enhancing the assembly of death-inducing signaling complex (DISC), which activates caspase-8 and the following cascade. Also, our unpublished data indicated that H. pylori enhances TRAIL-mediated apoptosis through dephosphorylating Akt, which is the upstream signaling of FLIPs. ER stress, a perturbation of ER function induced by accumulation of misfolded proteins, has been reported to be associated with H. pylori pathogenesis and that the crosstalk between apoptosis and ER stress has been previously reported. Moreover, ER stress is induced within human gastric epithelial cells during H. pylori infection, and the infection results in cell death. Moreover, ER stress inhibits the activity of mTORC2/Akt signal transduction through GSK-3β-mediated phosphorylation of Rictor. In this thesis, we investigated how H. pylori sensitized TRAIL-mediated apoptosis in human gastric epithelial cells through the perturbation of ER homeostasis. First, we analyzed the time course expression of two ER stress markers, GRP78 and CHOP. The results revealed the significant downregulation in GRP78 expression and increased level of CHOP within AGS cells in simultaneously co-cultured with H. pylori and treated with TRAIL. Moreover, the time course result demonstrated the degradation of GRP78 and FLIPs in AGS cells within 9 hours of H. pylori co-culture. Since the degradation of GRP78 implied the perturbation of ER function and activated the downstream unfolded protein response (UPR) signal transduction, we further examined the effect of ER stress induction on AGS cells by thapsigargin. The results revealed that induction of ER stress sensitized AGS cells to TRAIL-mediated apoptosis and downregulated FLIPs. We further investigated whether UPR blockage of ER stress signal transduction would reverse the H. pylori-sensitized, TRAIL-mediated apoptosis. Inhibition of eIF2α dephosphorylation by salubrinal could attenuate H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells. Furthermore, knockdown of eIF2α expression by siRNA attenuated H. pylori-sensitized, TRAIL-mediated apoptosis and restored the degradation of FLIPs and cleavage of caspase-8. Also, knockdown of Akt expression by siRNA enhanced eIF2α phosphorylation and downregulated FLIPs. Taken together, these results suggested that H. pylori sensitizes TRAIL-mediated apoptosis via Akt-eIF2α-FLIPs axis. Studying the role of ER stress in H. pylori-sensitized, TRAIL-mediated apoptosis provides candidates for potential therapeutic targets in H. pylori-associated diseases.

誌謝 i
中文摘要 ii
Abstract iv
Introduction 1
1. Helicobacter pylori 1
2. TRAIL-mediated apoptosis and H. pylori infection 1
3. DISC and FLIPs in TRAIL-mediated apoptosis 2
4. Akt pathway in regulation of FLIPs 2
5. ER stress in TRAIL-mediated apoptosis and H. pylori infection 3
Rationales 5
Materials and Methods 6
Results 16
1. TRAIL-mediated apoptosis in AGS cells is sensitized by H. pylori via FLIPs-Akt axis 16
2. ER stress positively corelated with TRAIL-mediated apoptosis in AGS cells 16
3. The inhibitor of eIF2α dephosphorylation restored FLIPs downregulation and attenuated H. pylori-sensitized TRAIL-mediated apoptosis in AGS cells 17
4. Overexpression of GRP78 failed to reverse H. pylori-sensitized, TRAIL-mediated apoptosis 18
5. Knockdown of eIF2α attenuated H. pylori-sensitized, TRAIL-mediated apoptosis 19
6. Knockdown of Akt enhanced the phosphorylation of eIF2α in human gastric epithelial cells 19
Discussion 21
1. Disruption of ER function in H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells 21
2. The effect of knockdown eIF2α in H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells 22
3. The interaction between Akt and eIF2α phosphorylation in H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells 24
Conclusions 26
References 27
Figures 31

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