背景與目的
幽門桿菌感染會引發慢性胃炎、胃潰瘍、胃腺癌，已被世界衛生組織認定為第一級致癌物。而胃裡的劉易斯B和唾液酸劉易斯X抗原則是扮演了幽門桿菌移植的受體。實驗室以前的研究發現，幽門桿菌感染的成年人比感染的兒童，在胃體部具有顯著性較高的細菌密度和劉易斯B強度。此外還發現，在小鼠中的胃唾液酸劉易斯X表現隨著年齡的增長而增加。另外研究揭示促分裂原活化蛋白激酶(MAPK)路徑可能調控了糖蛋白的合成。因此，本篇宗旨是想釐清在幽門桿菌感染之後是否促分裂原活化蛋白激酶路徑調控劉易斯抗原的表現，而造成了在小孩與成人之間不同的幽門桿菌移生密度。

結果
首先，我們的目的是確認在幽門桿菌感染的小孩及大人的原發性胃表皮細胞在感染之後是否有劉易斯抗原表現、幽門桿菌移生、細胞激素以及促分裂原活化蛋白激酶(MAPK)活性的差異的不同。所以利用人類胃胎兒上皮(HSFE)細胞和人類胃永生上皮(GES-1)初級胃表皮細胞來模擬小孩和成人的原發性胃上皮細胞。每一組別都會感染不同時間週期的幽門桿菌。結果顯示在成人相較於小孩的胃表皮細胞有顯著性較高而且較早的幽門桿菌移生強度表現。並且幽門桿菌誘導較高的劉易斯B表現強度和唾液酸劉易斯X分佈比例在成人胃表皮細胞，相較於小孩胃表皮細胞。而且在小孩胃表皮細胞，幽門桿菌誘導較高的IL-6 和 IL-8 表現，此外幽門桿菌誘導的MAPK活性在GES-1和HSFE是不一樣的。這些結果顯示劉易斯抗原調控的幽門桿菌移生，和細胞激素，MAPK反應一樣，在小孩與大人的胃裡是不同的。
第二，為了要確認MAPK訊號路徑對劉易斯抗原表現及幽門桿菌移生有何影響而利用了特定的MAPK單元的抑制劑及SiRNA敲除。結果顯示MAPK siRNA 轉染，導致降低了成人細胞的幽門桿菌移生密度，但是增加了小孩細胞的幽門桿菌移生密度，尤其是在轉染JNK siRNA的組別。 而且，利用JNK siRNA 確實也增加了GES-1的劉易斯B表現，但降低HSFE的劉易斯B和唾液酸劉易斯X抗原表現。 相同的結果也發現在磷酸化JNK抑制劑的實驗，而效果隨著劑量增加而增加。 這些結果說明JNK磷酸化活性會藉由調控劉易斯抗原表現使GES-1幽門桿菌移生密度增加，但是會降低在小孩細胞上。 並且，IHC染色結果發現幽門桿菌誘導的磷酸化JNK分數在成人胃的切片檢體顯著性的高於小孩。這臨床研究再次證實了幽門桿菌誘導的MAPK訊號的差異性在幽門桿菌感染的小孩與大人的胃檢體。
第三，我們探討前處理與後處理益生菌是否可以降低幽門桿菌誘導的MAPK訊號和幽門桿菌移生密度。結果發現，預先處理比菲德氏菌而不是乳酸桿菌會明顯降低JNK的磷酸化在GES-1和HSFE細胞上。 然而，在GES-1細胞上，預先處理比菲德氏菌和乳酸桿菌都會增加幽門桿菌移生密度在一小時及六小時。而對HSFE沒有幽門桿菌移生密度的影響。 此外，後處理比菲德氏菌和乳酸桿菌都會降低GES-1幽門桿菌移生密度，HSFE沒有。然而，在GES-1的抑制效果是藉由其他因子，而不是藉由調控路易斯抗原。

結論
幽門桿菌感染會誘導不同細胞激素、MAPK、劉易斯抗原表現在GES-1和HSFE上，這些不同可能導致感染後GES-1幽門桿菌密度會高於HSFE。而較高的磷酸化JNK活性可能貢獻了部分較高的劉易斯抗原表現和幽門桿菌密度在GES-1上，相較於HSFE。最後，雖然預先處理比菲德氏菌可以顯著性的降低JNK的磷酸化在GES-1和HSFE上，後處理，而不是預處理比菲德氏菌和乳酸桿菌可以抑制幽門桿菌移生在GES-1上。然而，抑制效果並不是藉由調控路易斯抗原。

Background and Purpose
Helicobacter pylori causes chronic gastritis, peptic ulcers, and adenocarcinoma and is classified as a group 1 carcinogen by the WHO. Gastric Lewis b (Leb) and sialyl‐Lewis x (sLex) antigens serve as the initiated and persistent receptors for H. pylori colonization, respectively. In our previous studies have shown that the H. pylori‐infected adults had significantly higher bacterial density and Leb intensity than infected children. Furthermore, the gastric sLex expressions in mice increased by age. Besides, studies have disclosed MAPK pathway may control glycoproteins synthesis. Therefore, we aim to validate whether the MAPK signal pathway mediate Lewis antigens expression after H. pylori infection and cause the different colonization density between children and adults.

Results
First, we aimed to identify whether H. pylori infection in child and adult primary gastric epithelium have different Lewis antigens expression, H. pylori colonization, cytokines production, and MAPK activity. Thus we applied “Human Stomach Fetal Epithelium (HSFE) cells” and “The SV40-immortalized human normal gastric epithelial cell line (GES-1)” to mimic “child” & “adult” primary gastric epithelium. Each group was challenged with H. pylori (MOI100) at various time periods. The results showed H. pylori colonization intensity was significantly higher and was earlier achieved full expression in GES-1 than in HSFE cells. Furthermore, H. pylori induced a higher Leb intensity and sLex percentage in GES-1 than in HSFE cells. The H. pylori-induced IL-6 and IL-8 expressions in HSFE cells were largely higher than in GES-1 cells. Moreover, H. pylori‐induced MAPK phosphorylation were different between HSFE and GES‐1. These results indicate that different Lewis antigen-mediated gastric H. pylori colonization, cytokines production, and MAPK responses between the stomach of children and adults.
Second, to identify the impacts of MAPK signaling pathway on Lewis antigen expressions and H. pylori colonization, the specific MAPK subunits’ inhibitors and SiRNA knockdown were used. The results showed that MAPK siRNA transfection decreased H. pylori colonization in GES-1, but increased in HSFE, particular in JNK siRNA knockdown. Using siRNA of JNK knockdown JNK protein decreased Leb expression in GES-1, but increased Leb and sLex expression in HSFE. The same results were shown in p-JNK inhibitor experiment with a dose-dependent manner. These results indicated that p-JNK activity increased H. pylori colonization in GES-1, but decreased in HSFE through regulating Lewis antigen expressions. Furthermore, H. pylori-induced p-JNK intensity scores were significantly higher in adults’ gastric biopsies than those from children by IHC staining. The clinical studies confirmed again that differences of H. pylori-induced MAPK signals between gastric samples biopsies from H. pylori-infected adults and children.
Third, we investigated whether pretreatment and posttreatment of probiotics could decrease H. pylori-induced MAPK activity and colonization density. The results found that pretreatment of B. latis but not L. acidophilus significantly reduced the JNK phosphorylation in both GES-1 and HSFE cells. However, pretreatment of both B. latis and L. acidophilus increased H. pylori colonization density at 1 and 6 hours in GES-1 but did not affect colonization density in HSFE cells. Furthermore, posttreatment of both B. latis and L. acidophilus decreased H. pylori colonization density in GES-1 but not HSFE. However, the inhibitory effect through factors other than regulating Lewis antigens in GES-1 cells.

Conclusion
H. pylori infection induced different cytokines, MAPKs, and Lewis antigens expression between GES-1 and HSEE cells, which may cause a higher H. pylori colonization density in GES-1 had more than HSFE. The higher JNK phosphorylation activity in GES-1 cells may contribute in part to the higher Lewis antigen and colonization density than in HSFE. Finally, although pretreatment of B. latis significantly reduced the JNK phosphorylation in both GES-1 and HSFE cells, posttreatment but not pretreatment of both B. latis and L. acidophilus could reduce H. pylori colonization in GES-1 cells. However, the inhibitory effect was not through regulating Lewis antigen expression.

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