Abstract
摘要:幽門螺旋桿菌感染已被確立為造成人類胃部之慢性發炎、胃潰瘍、十二指腸潰瘍之主要因素,並 且與胃癌有高度的相關性。雖然有許多幽門螺旋桿菌的毒性因子被發現,但其致病機制至今仍尚未完全釐清。幽門螺旋桿菌感染所引起的發炎反應是腸胃道疾病的主因,而本實驗室先前發現與胃癌相關的幽門螺旋桿菌新毒性因子HSP10,會使人類胃癌細胞及人類周邊血液單核細胞釋放前發炎細胞激素。此外相對於胃炎及十二指腸潰瘍之病人,HSP10對胃癌病人血清具有很高的抗原性,顯示HSP10可能經由引起發炎反應在胃癌發生過程中扮演重要角色。HSP10對於胃癌之發生可能具有相當重要角色,本研究希望進一步探討HSP10引起發炎反應之機制。經由序列比對後發現幽門螺旋桿菌HSP10除了具高保留性的A domain還比其他種細菌多出一段獨有的B domain,有可能與造成細胞發炎有關。於是我們想知道A domain或B domain單獨是否具有引起發炎反應的能力。同時以細胞實驗探討其訊息傳遞路徑,希望找出HSP10是經由何種免疫受器來啟動下游訊息傳遞。接著利用免疫沉澱、共軛焦顯微鏡及表面電漿共振等方法來確認HSP10與其免疫受器之間的交互作用。透過表面電漿共振法我們還想探討HSP10是否可能藉由其高度帶電之B domain與細胞膜接合進而促進其與免疫受器的結合。此外我們也會利用點突變分析來找出B domain中與引起發炎反應相關之重要胺基酸,接著利用圓二色光譜與表面電漿共振來判別這些胺基酸是否造成構形改變而影響到受器之辨識。最後利用小角度散射方法探討HSP10重要胺基酸對其結構之影響。這些研究對於幽門螺旋桿菌與宿主間的相關性有更清楚的了解,並期待對發展胃癌治療的新方向以及藥物開發有所貢獻。
Abstract: Helicobacter pylori infection has been proven as a major cause of chronic gastric inflammation, gastric ulcer, duodenal ulcer, and is highly related to gastric cancer. Although many virulence factors of H. pylori have been reported, the pathogenic mechanism associates H. pylori and gastric diseases still remains unclear. Chronic inflammation induced by H. pylori colonization is considered as a main cause of gastric diseases. HSP10 protein secreted by H. pylori was defined as a novel gastric cancer-associated virulence factor in our previous study. We found that HSP10 can induce proinflammatory cytokines releasing in both human gastric carcinoma cells and peripheral blood mononuclear cells (PBMCs). We also found in comparison with gastritis and duodenal ulcer patients’ sera, HSP10 showed a much higher seropositivity of gastric cancer. These finding indicate that HSP10 may play an important role in gastric carcinogenesis via induction of inflammatory responses.Considering the association between HSP10 and gastric cancer may have an important role in H. pylori pathogenesis, we would like to study the mechanism of proinflammatory cytokines releasing in gastric carcinoma cells induced by HSP10. Sequence alignment of H. pylori HSP10 with other bacterial HSP10s showed that H. pylori HSP10 protein has highly conserved chaperonin domain (domain A) and an additional 28 amino acids at their C-termini (domain B) which may contribute to inflammatory responses. We want to examine whether domain A or domain B only have immune-stimulation ability both in vitro and in vivo. Next, we would like to examine the signaling pathway involved in HSP10 induced immune responses. We hope to identify the molecules involved in the signaling pathway, and also the immune receptor responsible for HSP10 induced signaling. After knowing HSP10 is recognized by which receptor, we would like to use confocal microscopy, co-immunoprecipitation and surface plasmon resonance (SPR) method to further confirm the interaction between HSP10 and the receptor. Considering domain B is unique to H. pylori HSP10 and contains highly-charged residues, we further use SPR to study the interaction between domain B and cell membrane which may contribute to facilitating HSP10 delivery to the receptor. In addition, we would like to find the crucial residues in HSP10 domain B for receptor recognition and for membrane binding by alanine scanning site-directed mutagenesis. After knowing which residues are important for cytokine induction, we can use CD spectrum and SPR method to find out which crucial residues contributing conformation change and receptor binding. Finally, we would like to use SAXS to understand the structural differences between HSP10 and functional inactive mutant. These findings will be important in the understanding of the host-pathogen interactions and in further contribute to new therapeutic strategies and drug development in gastric cancer treatment.
Keyword(s)
幽門螺旋桿菌
胃癌
發炎反應
毒性因子
熱休克性蛋白
免疫接受器
蛋白質與細胞膜結合
Helicobacter pylori
gastric cancer
inflammatory response
virulence factor
heat shock protein
immune receptor
lipid-protein interaction