利用磷酸化蛋白質體學技術分析幽門螺旋桿菌感染胃腺癌上皮細胞誘發蛋白質磷酸化修飾變異

在過去的研究中發現，幽門螺旋桿菌的感染可能引起多種消化道疾病如胃炎、潰瘍，甚至可能會導致胃癌的發生。當幽門螺旋桿菌感染宿主細胞時，會產生的宿主反應有細胞形態的改變、細胞增生、發炎或是細胞凋亡等。由於幽門螺旋桿菌與消化道疾病的顯著關連性，多年來不斷地有研究嘗試去瞭解受到感染的宿主細胞中訊息傳遞路徑的調控。由於蛋白質磷酸化快速而可逆的調控特性，能夠更即時地反應細胞受到刺激時所產生的訊息傳遞情形，也因此本實驗嘗試利用近年來方興未艾的磷酸化蛋白質體學技術，以研究當宿主細胞被幽門螺旋桿菌感染時，細胞內所誘發的蛋白質磷酸化變異，並探討可能代表的生理意義。經過磷酸化純化的工具增加磷酸化蛋白的比例，接著再透過質譜鑑定的方式來分析蛋白質磷酸化的情形，並將特定在幽門螺旋桿菌的感染後會被磷酸化的蛋白質種類利用訊息傳遞資料庫的分析將鑑定結果推測出可能的訊息傳遞網路。另外，也應用質譜定量技術來精確地比較感染前後磷酸化情形的差異。這些被鑑定到的磷酸化蛋白質參與在細胞的生長與增殖、維持細胞形態、細胞內與細胞間訊息傳遞、細胞凋亡等生理反應。透過西方墨點染色法的驗證以及過去文獻的探討，顯示幽門螺旋桿菌的感染確實會透過磷酸化調控的方式，來影響Akt和glycogen synthase kinase 3b的訊息傳遞路徑，並且這些變異多是指向促進細胞的增殖。而其他如形態改變、發炎反應等也都有磷酸化調控的參與。未來可望針對這些結果做更進一步的驗證以探討其生理意義，並以這些資訊來闡明幽門螺旋桿菌的致病機制。

關鍵字

幽門螺旋桿菌；胃癌；磷酸化蛋白質體學；訊息傳遞

並列摘要

It has been found that Helicobacter pylori infection could result in a variety of gastroduodenal diseases such as gastritis, ulcer, and even gastric cancer. When H. pylori infection occurs, different kinds of host responses such as morphology changes, proliferation, inflammation, and apoptosis would be induced. Because of this significant relationship between H. pylori and gastroduodenal diseases, many researches have focused on the regulation of signal transduction in host cells during H. pylori infection. Due to fast and reversible regulation, signal transduction through protein phosphorylation in host cells can react to stimulation in a real-time fashion. As a result, phosphproteomics tools were used to identify the phosphorylation regulation changes in host cell during H. pylori infection. After phosphoprotein enrichment and LC/MS-MS identification, the phosphorylated proteins specific in H. pylori infected host cells were analysed by the signal transduction database tool. In addition, quantitative technique of mass spectrometry was also used to accurately compare the differences of phosphorylation induced by infection. These identified phosphoproteins involve in cellular growth and proliferation, cell morphology, in-cell and cell-to-cell signaling and apoptosis. According to western blot validation and literature searches, it is found that H. pylori would induce phosphorylation of Akt and glycogen synthase kinase 3b, which might involve in cell proliferation. Besides, morphology changes and inflammation would also be induced through phosphorylation regulations of other proteins. However, the phosphorylation regulations and their physiological roles need to be further validated. This information may help to demonstrate more comprehensive pathogenic mechanisms of H. pylori infection.

並列關鍵字

Helicobacter pylori ； gastric cancer ； phosphproteomics ； signal transduction

參考文獻

1. Marshall, B.J., et al., Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, 1984. 1(8390): p. 1311-5.

2. Ernst, P.B, et al., The disease spectrum of Helicobacter pylori: the immunopathogenesis of gastroduodenal ulcer and gastric cancer. Annu. Rev. Microbiol., 2000. 54: p. 615-40.

3. Everhart, J.E., Recent developments in the epidemiology of Helicobacter pylori. Gastroenterol Clin. North. Am., 2000. 29(3): p. 559-78.

4. Miyaji, H., et al., Helicobacter pylori infection occurs via close contact with infected individuals in early childhood. J. Gastroenterol. Hepatol., 2000. 15(3): p. 257-62.

5. Lauren, P., The Two Histological Main Types of Gastric Carcinoma: Diffuse and So-Called Intestinal-Type Carcinoma. An Attempt at a Histo-Clinical Classification. Acta. Pathol. Microbiol. Scand., 1965. 64: p. 31-49.

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利用磷酸化蛋白質體學技術分析幽門螺旋桿菌感染胃腺癌上皮細胞誘發蛋白質磷酸化修飾變異

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