幽門螺旋桿菌 GroES 蛋白在試管中及活體內誘發前發炎細胞 激素釋放

Abstract

過去的研究中，幽門螺旋桿菌已被證實會造成胃部的發炎，並且與許多胃部的疾病(胃炎、胃潰瘍、十二指腸潰瘍、胃癌等)有高度的關連性，在本實驗室先前的研究中，發現了幽門螺旋桿菌新的致病因子 GroES 蛋白，幽門螺旋桿菌的 GroES 蛋白會使得人類周邊血液單核 細胞釋放前發炎細胞激素(proinflammatory cytokines)，這個現象可能與胃癌的形成有關。然而，相較於幽門螺旋桿菌的 GroES 蛋白，大腸桿菌的 GroES 蛋白並不會造成周邊血液單核細胞產生前發炎細胞激素，在比對幽門螺旋桿菌與其他微生物的 GroES 蛋白序列後，發現幽門螺旋桿菌的 GroES 蛋白在羧基端有 28 個胺基酸片段的延伸。有鑑於此，我們想探討此一片段對於幽門螺旋桿菌的致病性是否扮演重要的角色。我們表現全長之幽門螺旋桿菌 GroES 蛋白(H. p GroES)、刪除羧基端 28 個胺基酸之 H. p ΔGroES(1-90)蛋白，以及大腸桿菌之 GroES蛋白(E. coli GroES)，並以試管內(In vitro)以及活體中(In vivo)實驗方法來探討這些蛋白的功能。在試管內的方法中，將三種 GroES 蛋白分別處理細胞後發現，不論是在人類周邊血液單核球、MKN45 細胞或是 THP-1 細胞中，都可以發現全長之 H.p GroES 蛋白會引發這些細胞高量的前發炎細胞激素介白素-8(Interkeukin 8, IL-8)表現，反觀經 H. p ΔGroES(1-90)以及 E.coli GroES 處理的組別，其介白素-8 的表現較 H.p GroES 蛋白處理的組別低。在活體中的方法中，我們使用斑馬魚當做模式生物，分別將這些種蛋白處理斑馬魚後，以定量聚合酶連鎖反應(Q-PCR)檢測這些組別其前發炎細胞激素的 mRNA 表現，結果顯示 H. p GroES 蛋白處理的組別中，其前發炎細胞激素介白素-1、介白素-8 相較其他蛋白處理組別有高量表現。接著使用原位核酸雜交法(In Situ Hybridization)染色觀察不同組別的切片後更進一步發現，H. p GroES 蛋白處理的組別，其前發炎細胞激素介白素-1、介白素-8 與腫瘤壞死因子 α(Tumor necrosis factorα, TNFα)RNA 高量表現於斑馬魚之消化系統區域，而在其他蛋白處理組別並無發現這樣的情況。綜合以上，幽門螺旋桿菌羧基端的 28 個胺基酸對於 H. p GroES 蛋白誘發前發炎細胞激素釋放扮演著重要的角色，也暗示 H. p GroES 蛋白，可能使胃上皮細胞及免疫細胞引發持續性發炎的情況。

Helicobacter pylori has been proven to cause stomach inflammation and many gastric diseases are highly related with H. pylori. In our previous study, we discovered a new H. pylori virulence factor—GroES protein. H. pylori GroES protein induces the peripheral blood mononuclear cells (PBMCs) release proinflammatory cytokines may be related to the formation of gastric cancer. However, E. coli GroES protein does not result in this phenomenon. Compared H. pylori with other microorganisms’ GroES protein sequence, revealed that the H. pylori GroES have the carboxyl extension of 28 amino acid fragment, and this fragment is unique to H. pylori was not found in other organisms. In view of this, it is necessary to disscuss how this fragment playing an important role in the pathogenicity of H. pylori. Recombinant proteins of full length H. p GroES (H. p GroES), removing the carboxyl 28 amino acids of the H. p GroES (H. p ΔGroES(1-90)), and E. coli GroES were prepared, using in vitro and in vivo methods to study function of these proteins. In in vitro methods, human PBMCs, MKN45 cells and THP-1 cells, had treated with three types of GroES protein, found that H. p GroES will lead to high levels of proinflammatory cytokine IL-8. In the cantrast, H. p ΔGroES(1-90) and E. coli GroES treatment groups had lower ability to induce IL-8 secretion in these cells. In in vivo approach, zebrafish were used as a model organism, dealing with these kinds of proteins in zebrafish. Using Q-PCR to analyze these groups of their mRNA expression showed that IL-1, IL-8 had high levels in H. p GroES treatment groups compared to control and other treatment groups. Performing In situ Hybridization to stain different groups of biopsy also discovered IL-1, IL-8 and TNF-α had higher RNA expression level at the digestive tract in zebrafish treated by H. p GroES. This phenomenon cannot be found in other treatment group. In summary, H. pylori Carboxyl 28 amino acids plays a key part in H. p GroES to induce the release of proinflammatory cytokines, and these results also suggested that H. pylori GroES protein may promote the gastric epithelial cells and immune cells inflammatory response.