胃幽門螺旋桿菌感染造成免疫組織相容性複合體相關抗原呈現系統被丙型干擾素刺激後阻斷作用的探討

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研究生：	黃昆隆 Huang, Kun-Long
論文名稱：	胃幽門螺旋桿菌感染造成免疫組織相容性複合體相關抗原呈現系統被丙型干擾素刺激後阻斷作用的探討
指導教授：	張鑑中 Chang, Chien-Chung
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	55
相關次數：	點閱：3 下載：0
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Helicobacter pylori is a gram-negative anaerobic bacterium which causes gastric ulcers in the stomach. A characteristic pathogenesis feature of H. pylori-caused gastric ulcers is vacuolation of gastric epithelial cells, which is associated with unresolved chronic inflammation in the course of the disease. There are many H. pylori virulence factors, such as VacA and CagA. These virulence factors may be responsible for immune suppression by down-regulating the expression of antigen presentation components, or changing the distribution of the MHC class I and class II molecules through altering intracellular vesicle trafficking. In our study, we have found antigen processing machinery (APM) components TAP1 and TAP2 were down-regulated both at the transcriptional and translational levels along with down-regulation of STAT promoter activity and of the IFN-□ responsive genes IRF-1 and GBP2. TAP1 and TAP2 down-regulation was corrected by a VacA inhibitor NPPB, suggesting the role of vacuolation in H. pylori-induced TAP down-regulation. Interestingly, both the level and distribution of MHC class II molecules were disrupted in H. pylori-infected HeLa cells. These MHC class II molecules were found to aggregate within H. pylori-induced vacuoles, where proteolytic processing was interrupted. These results and the co-localization of MHC class II molecules and their chaperon DM within H. pylori-induced vacuoles imply the presence of unstable, peptide-free MHC class II molecules which were transported to the cell surface poorly. Taken together, our results indicate that both MHC class I and MHC class II presenting antigen machinery were disrupted by H. pylori, thus impairing the presentation of endogenous peptides and exogenous antigens to CD8+ and CD4+ T cells. These findings may explain why H. pylori can escape host immune attack and may suggest strategies to treat gastric diseases induced by H. pylori.

胃幽門螺旋桿菌是一種在胃部造成胃潰瘍的格蘭氏陰性菌。胃幽門螺旋桿菌造成胃潰瘍的發病特徵是讓胃表皮細胞空泡化，這也跟此疾病無法解決的慢性發炎有關。胃幽門螺旋桿菌有許多致病因子就像是VacA和CagA。這些致病因子可能負責參與免疫逃避藉由降低調控組織相容性複合體第一型抗原呈現構成分子的表現或著藉由改變免疫組織相容性複合體第二型的分佈透過細胞內的囊泡運輸。在我們的研究中，受胃幽門螺旋桿菌感染的上皮細胞中抗原呈現構成分子中的TAP1和TAP2在轉錄和轉譯層面被明顯的下調。然而在受胃幽門螺旋桿菌感染的上皮細胞中，大部分受丙型干擾素刺激的基因都會被胃幽門螺旋桿菌下調藉由降低STAT啟動子的活性。這種TAP1和TAP2的下調作用卻會被VacA的抑制劑NPPB給回復回來。此外，組織相容性複合體第二型的分佈也會受到胃幽門螺旋桿菌的干擾作用而聚集在細胞內的囊泡中。這暗示沒有胜肽可以嵌到免疫組織相容性複合體第二型中，空的免疫組織相容性複合體第二型是不穩定的且很難被送到細胞表面。總結，免疫組織相容性複合體第一型和第二型兩者的抗原呈現機制都被胃幽門螺旋桿菌干擾，所以不管內源性或著胞吞的胜肽都很難被呈現到細胞表面去刺激CD4+或CD8+淋巴細胞。我們推測這是胃幽門螺旋桿菌逃避寄主免疫攻擊的機制之一。藉由清楚了解這些機制，我們可以找出防止胃幽門螺旋桿菌造成的胃疾方法。

謝誌……………………….............................................................................................I
Abstract………………………………………………………………………………..II
中文摘要……………………………………………………………………………..IV
Contents…………………………………………………………………………….....V
List of Table………………………………………………………………...…...….VIII
List of Figure……………………………………………………………………….VIII
Chapter 1 Background and Significace………………………………………………..1
1.1    Introduction of Helicobacter pylori………………………………………….1
1.2    Vacuolating Cytotoxin A (VacA)……………………………………………3
1.3    MHC-associated Antigen Presentation…………………………………...….4
1.4    Hypothesis and Specific Aims……………………………………………….6
Chapter 2 Materials and Methods……………………………………………………..8
    2.1 Cell Culture…………………………………………………………………..8
    2.2 Bacterial Strains and Infection……………………………………………….8
    2.3 Staining of HeLa Cells with Neutral Red…………………………………….9
    2.4 Antibodies……………………………………………………………………9
    2.5 Plasmids…………………………………………………………………….10
    2.6 Reverse Transcription (RT)-PCR…………………………………………...11
    2.7 Real Time-PCR……………………………………………………………..11
    2.8 Flow Cytometry……………………………………………………………..12
    2.9 Western Blotting……………………………………………………………12
    2.10 Immunofluorescence Staining…………………………………………….13
    2.11 Luciferase Assay…………………………………………………………..14
Chapter 3 Results…………………………………………………………………….15
    3.1 Induction of HeLa Cell Vacuolation by Helicobater pylori………………...15
    3.2 Down-regulation of TAP1 and TAP2 Protein Expression in HeLa Cells Infected by H. pylori in the Presence of IFN-□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□
□□□□□□□□3.3 Down-regulation of TAP1 and TAP2 Protein Expression in AGS Cells infected by H. pylori in the Presence of IFN-□ ……………..………………16
□□□□Down-regulation of TAP1 and TAP2 mRNA Expression in HeLa Cells Infected by H. pylori in the Presence of IFN-□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□
□□□□□□□□□□□ TAP1 and TAP2 Protein and mRNA Expression were not Down-regulated in HeLa Cells Incubated with H. pylori Lysates……………………………….18
    3.6 Downregulation of TAP1 and TAP2 Promoter Activity in HeLa cells Infected by H. pylori ………………………………………………………………...19
    3.7 Downregulation of IFN-□□Responsiveness in HeLa Cells Infected by H. pylori as Indicated by Reduced STAT Promoter Activity and Reduced Expression of IFN-□ Responsive genes IRF-1 and GBP2..............................20
3.8 Restoration of TAP1 and TAP2 mRNA Expression in H. pylori –infected HeLa Cells Treated with the V-type ATPase Inhibitor NPPB.........................20
    3.9 Clustering of the MHC class II Molecules in the Cytosol and Reducing amount of MHC Class II Molecules on Cell Surface in H. pylor–infected HeLa cells.......................................................................................................21
Chapter 4 Conclusions and Discussion………………………………………………23
    4.1 Conclusions…………………………………………………………………23
    4.2 Discussion…………………………………………………………………..24
Chapter 5 Future Directions………………………………………………………….27
Referances…………………………………………………………………...……….29
Tables………………………………………………………………………………...32
Figures………………………………………………………………………………..34













Tables………………………………………………………………………………..32
Table 1. RT-PCR Primers List……………………………………………………….32
Table 2. Real-Time PCR primers list………………………………………………...33
Figures………………………………………………………………………………34
Figure 1. The MHC Class I Pathway of Processing of Cytosolic Antigens………….34
Figure 2. The MHC Class II Pathway of Processing Internalized Vesicular Antigens……………………………………………………………………………...35
Figure 3. TAP1/2 Promoter-driven Reporter Constructs……………………………..36
Figure 4. The HeLa Cell Vacuolation Induced by H. pylori…………………………37
Figure 5. The down-regulation of TAP1 and TAP2 Protein Expression in H. pylori-infected HeLa Cells in the Presence of IFN-□………………………………...39
Figure 6. The down-regulation of TAP1 and TAP2 protein expression in H. pylori-infected AGS cells in the presence of IFN-□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□
Figure 7. The Down-regulation of TAP1 and TAP2 mRNA Expression in H. pylori-infected HeLa Cells in the Presence of IFN-□……………………………...…42
Figure 8. The Down-regulation of TAP1 and TAP2 mRNA Expression Quantified by Real-time PCR in H. pylori-infected HeLa Cells…………………………………….44
Figure 9. Both mRNA and Protein Level of TAP1 and TAP2 were not Down-regulated in HeLa Cells Treated with H. pylori Lysates………………………………………..45
Figure 10. H. pylori Factors Reduced the Activity of TAP1, TAP2, and STAT-responsive Promoter in HeLa Cells Treated with IFN-□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□
Figure 11. The IFN-□□Responsive Genes, IRF-1 and GBP2, were Down-regulated in H. pylori-infected HeLa Cells in the Presence of IFN-□………………………………...48
Figure 12. The Down-regulation of TAP1 and TAP2 mRNA Expression was Restored by NPPB in the H. pylori-infected HeLa Cells………………………………………49
Figure 13. The Amount of Cell Surface MHC Class II Molecules was Reduced in H. pylori-infected HeLa Cells…………………………………………………………...51
Figure 14. MHC Class II Molecules were Clustered in the Cytosol in H. pylori-infected HeLa Cells in the Presence of IFN-□………………………………...52
Figure 15. DM and Ii were Clustered with MHC class II in the Cytosol in H. pylori-infected HeLa Cells in the Presence of IFN-□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□

                                

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