幽门螺杆菌临床分离株CagA的序列比对及其对胃上皮细胞形态与功能的影响

Sequence of Helicobacter pylori clinical isolate CagA and its effect on the morphology and function of gastric epithelial cells

摘要:

【背景】细胞毒素相关基因A蛋白(Cytotoxin Associated Gene A protein，CagA)是幽门螺杆菌(Helicobacter pylori)重要的效应蛋白，CagA的多态性与胃癌的发生发展密切相关。【目的】比较幽门螺杆菌临床分离株的CagA结构差异，探讨不同CagA对胃上皮细胞形态及功能的影响。【方法】对27株幽门螺杆菌的CagA序列进行比对，分析氨基酸组成差异及变异情况，用含不同CagA序列的5株H. pylori感染低恶性胃上皮细胞AGS 6 h，感染复数(Multiplicity of infection，MOI)为30:1，显微镜观察细胞形态变化，Western blot法检测极性调节激酶1b (Polarity-regulating kinase 1b，PAR1b)的表达，ELISA检测培养液中白介素8 (Interleukin-8，IL-8)的浓度。【结果】临床分离株的CagA存在结构和氨基酸组成差异，西方株的EPIYA基序存在更多变异，具有完整cagA基因的菌株感染后细胞形态发生显著变化。Western blot分析结果显示：与对照组比较，西方株NCTC 11639和H. pylori 26695感染的细胞中PAR1b的表达升高，而东亚株H. pylori GZ7感染的细胞中PAR1b表达降低，差异均有统计学意义(P<0.05)；突变株H. pylori GZ15及H. pylori GZ7/ΔcagA感染后PAR1b的表达无显著变化。与对照组比较，实验组中H. pylor感染的细胞培养液中IL-8浓度均增加，东亚株促进IL-8分泌的能力大于西方株。【结论】含不同CagA的幽门螺杆菌发挥不同的生物学功能。东亚株能抑制PAR1b的表达，促进IL-8分泌的能力更强，H. pylori感染引起的细胞形态变化依赖cagA基因的完整性。

Abstract:

[Background] Cytotoxin associated gene A protein (CagA) is one of the most important effectors of Helicobacter pylori, and its polymorphism is suggested to be involved in the development and progression of gastric carcinoma. [Objective] To compare the structural differences of CagA as an H. pylori clinical isolate, and explore its effects on the morphology and function of gastric epithelial cells. [Methods] A total of 27 sequences of CagA were analyzed to reveal the differences in composition and amino acids variation. Five H. pylori strains containing different CagA sequences were used to infect low malignant gastric epithelial AGS cells for 6 hours at a multiplicity of infection (MOI) of 30. Then the cell morphology was observed by the microscope and the expression of polarity-regulating kinase 1b (PAR1b) was determined by the Western Blot. Finally, the concentration of interleukin-8 (IL-8) in the medium was detected using enzyme-linked immunosorbent assay (ELISA). [Results] It was found that there were differences in the structure and composition of amino acid of CagA. In particular, the EPIYA motifs of Western strains was detected with more variation, and the cell morphology of the strain with the intact CagA gene changed significantly after infection. Compared with the control group, the expression of PAR1b in the AGS cells infected with Western strains of NCTC 11639 and 26695 increased significantly, while expression of PAR1b in the cells infected with East Asian strain GZ7 decreased significantly, and both of the differences were statistically significant (P<0.05). However, there was no significant difference in the expression of PAR1b in the AGS cells infected with H. pylori GZ15 and H. pylori GZ7/ΔcagA. The concentration of IL-8 in the experimental group was higher than that in the control group for East Asia and Western strains (P<0.05). Furthermore, the potential of East Asia strains to promote IL-8 secretion was greater than that of Western strains. [Conclusion] H. pylori containing different CagA performs different biological functions. The East Asian strains can inhibit the expression of PAR1b and have a more vital ability to promote the secretion of IL-8. In addition, the changes in cell morphology caused by H. pylori depend on the integrity of the cagA.