- 學位論文
胃幽門螺旋桿菌 CagA 分子在細胞內的分布與誘導 B 細胞活化/分化的角色之研究
Study of H. pylori CagA intracellular localization and induction of B cell activation/differentiation
摘要
胃幽門螺旋桿菌 (Helicobacter pylori) 首先在患有胃炎以及胃潰瘍的病患胃部所發現，其可造成慢性胃炎、胃潰瘍、胃黏膜相關淋巴組織淋巴癌以及胃癌等疾病，在胃幽門螺旋桿菌所產生的蛋白中，CagA 被認為是主要的毒性蛋白，而CagA 是一個 125-145 kDa 的蛋白質，當胃幽門螺旋桿菌附著上胃上皮細胞後，利用編碼在 Cag PAI 上的第四型分泌系統 (type VI secretion system) 或其他分子送入到胃上皮細胞中，進一步被細胞中的磷酸酶 Src family kinase 和 c-Abl磷酸酶磷酸化，形成 phospho-CagA 和 SHP-2 結合，進而活化 SHP-2，從而改變細胞與細胞間的附著、細胞型態的改變以及細胞的遷移。SHP-2 不正常持續的活化情形也和癌症的生成有關，CagA 在胃上皮細胞內會加速細胞週期，刺激細胞增生。另一方面，具有CagA基因的胃幽門螺旋桿菌也與 low-grade gastritis mucosa associated lymphoid tissue (MALT) lymphoma 的形成有關，之前實驗室所發表的論文也證明 CagA 可以經由第四型分泌系統送入 B 細胞中並活化 MAP kinase 分子─ERK 與 P38，以及增加抗細胞凋亡分子 BCL-2 和 BCL-XL 的表現，在 MALT lymphoma 生成中，CagA 和 B 細胞的直接作用可能扮演抗細胞凋亡並促進細胞增生的角色。 目前對於 CagA在細胞內的位置及作用仍不清楚，我們進一步探討 CagA進入細胞後的分布以及磷酸化對其影響。我們以 B 細胞和胃幽門螺旋桿菌共同培養並且使用核質分離的技術，觀察到 CagA 確實可存在於細胞質和細胞核中，而且隨時間增加進入核內的 CagA 有增加的現象，細胞核內的 CagA 或是phospho-CagA 較細胞質能有更持續的表現，此現象可能代表 CagA 在核內的具有其生理意義進而影響細胞的生存或活化。接著我們再製作不同 N 端 truncate 片段的 CagA 質體 DNA 轉染進 AGS 細胞中，觀察 CagA 進核的方式，我們發現到 N 端 600-892 a.a.對於 CagA 進入細胞核有重要的角色。在 B 細胞分化上，我們觀察到胃幽門螺旋桿菌可使 B 細胞內 BCL-6 蛋白質表現量下降，然而在CagA 基因剔除的菌株中，BCL-6 並無明顯下降。利用 Real-time PCR 測量 BCL-6 mRNA 表現，也發現 BCL-6 mRNA 表現受到抑制，但是在 CagA 基因剔除菌株共同培養的組別並未發現相同結果。與漿細胞分化相關的轉錄分子 BLIMP-1，利用 Western blot 以及 Real-time PCR 我們也觀察到和BCL-6相反的表現，蛋白量和mRNA 都有明顯提升，在 CagA 基因剔除菌株共同培養的組別則無此現象。這些結果顯示 CagA 被胃幽門螺旋桿菌送入 B 細胞可以使 BCL-6表現下降，BLIMP-1表現上升。我們的研究結果顯示這些分子的表現可能和 CagA在 B 細胞內的位置有關並可能參與胃幽門螺旋桿菌造成胃黏膜相關淋巴組織淋巴瘤的形成。期待未來的研究結果能進一步釐清 CagA 誘導 B 淋巴細胞的活化或 MALT lymphoma 的形成有重要的啟發與貢獻。
並列摘要
Abstract Helicobacter pylori was first discovered in the stomach of patients with gastritis and gastric ulcer and is linked to the development of chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. In proteins produced by Helicobacter pylori, CagA is considered as the major virulent protein about 125-145 kDa and can be injected through T4SS (type four secretion system) and other molecules encoded on CagPAI into gastric epithelial cell when Helicobacter pylori contact with gastric epithelial cell. The CagA protein in gastric epithelial cell is further phosphorylated by Src family kinase and c-Abl. Phospho-CagA binds with SHP-2 and activates SHP-2 in a phosphorylation dependent manner. Activated SHP-2 can change actin filament structure resulting in cell-cell adhesion, cell phenotype and migration alternation. SHP-2 deregulation associates with cancer formation. CagA in gastric epithelial cell could accelerate cell cycle and stimulate cell proliferation. On the other hand, CagA+ Helicobacter pylori is associates with low-grade gastritis mucosa-associated lymphoid tissue (MALT) lymphoma formation. Previous study showed that CagA can be injected through T4SS into B cells and activate MAP kinase─ERK and P38 along with increased anti-apoptosis molecule BCL-2 and BCL-XL expression. In MALT lymphoma genesis, CagA directly interacts with B cells may contribute to anti-apoptosis and promoting proliferation in B cells. By now, CagA intracellular localization and function are currently unknown. We further investigated distribution of translocated CagA in cells and phosphorylation effect on distribution. So far, we co-cultured B cells with H. pylori then applied to Nucleocytoplasmic separation and observed that CagA localize in cytosol and nucleus and nucleus CagA persist longer than cytosol CagA. CagA nucleus localization may represent some biological mechanism so as to affecting B cells survival or activation. Next, we use N-terminal truncate CagA transfected into AGS cells study which sequence or motif is required for translocation and we find that there is no significant difference in N-terminal truncate CagA except lacking CagA 600-892 a.a.. In B cell differentiation, we also found that H. pylori decreases BCL-6 protein expression, but BCL-6 does not decrease when BJAB cells co-cultures with CagA knock-out strain. Using Real-time PCR to investigate BCL-6 mRNA expression, we find that expression of BCL-6 mRNA is inhibited, but not in BJAB cells co-cultures with CagA knock-out strain. We used Western blot and Real-time PCR to explore whether H. pylori can affect the expression of plasma cell differentiation associated molecule and find that in contrast to BCL-6, BLIMP-1 protein and mRNA increase in co-culture with H. pylori but not with CagA knock-out strain. Our research results indicate that CagA in BJAB cells down-regulate BCL-6 and up-regulate BLIMP-1 which expression may associate with CagA localization in BJAB cells and lead to MALT lymphoma formation. Our results in this study may provide an inspiration and contribution on CagA-induced B cells activation/differentiation or MALT lymphoma genesis.