幽门螺杆菌毒素相关蛋白A的EPIYA多态性对胃上皮细胞AGS形态及IL-8表达的影响

Effects of the Patterns of Helicobacter pylori Cag A EPIYA Motifs on AGF Cell Morphology and IL-8 Secretion Levels

目的:比较研究幽门螺杆菌毒素相关蛋白A(Cag A)不同分型对胃上皮细胞AGS形态及IL-8表达影响的差异。方法:设计、人工合成并优化不同cag A基因型,构建相应表达载体,转染胃上皮细胞AGS后观察不同型Cag A对细胞形态及IL-8表达的影响。结果:转染后每6 h进行细胞形态观察,于24、36 h对形成蜂鸟状细胞的数量进行统计分析,结果表明,各型Cag A均能引起发生蜂鸟状改变的细胞数量增加;较空载体对照,Cag A的6种型别皆有非常显著差异,Cag A ABCCC与其余5种Cag A型别有非常显著差异。同时于转染后12、36 h对细胞分泌的IL-8进行检测,统计分析表明,转染后12 h,转染各型Cag A细胞的IL-8表达量开始出现差异;转染后36 h,较空载体对照,Cag AABD、Cag A J-Western、Cag A ABDD和Cag A ABCCC存在非常显著差异,Cag A ABDD和Cag A ABCCC分别与Cag AABD、Cag A ABC存在非常显著差异。结论:幽门螺杆菌Cag A可引起转染后的AGS细胞发生蜂鸟状改变及IL-8表达增加,EPIYA基序C数目的增加与细胞蜂鸟状改变呈正相关,EPIYA基序C和D数目的增加与IL-8表达呈正相关。

Objective：To investigate the effects of the patterns of EPIYA motifs of Helicobacter pylori cytotoxin-associated gene A（Cag A） on the cell morphology and the IL-8 secretion levels of AGS cells.Methods：The differ-ent genotypes of EPIYA motifs of cag A were designed and the codons were humanized,then the synthetic frag-ments were assembled and constructed into pc DNA3.1 vector.The recombinants were transfected into AGS cellsand the effects of patterns of EPIYA motifs of Cag A on cell morphology and the IL-8 secretion levels were ob-served.Results：Cellular morphological observation was conducted every 6 h after the transfection,and the num-bers of hummingbird-like cells were counted at 24 and 36 h respectively.The statistical results showed that allCag A genotypes could increase the number of cells with hummingbird-like change.Compared with the mock-vehi-cle controls,all the six Cag A types showed a remarkably significant differences;compared with Cag A ABCCC,thefive Cag A types left showed a remarkably significant differences.In addition,IL-8 secreted by these cells 12 and36 h after transfection was detected respectively.The statistical analysis revealed that,at 12 h after transfection,the secretion of IL-8 began to appear difference among the six Cag A types;at 36 h after transfection,comparedwith the mock-vehicle controls,Cag A ABD,Cag A J-Western,Cag A ABDD and Cag A ABCCC showed a remark-ably significant differences.Although both Cag A ABD and Cag A ABDD belonged to East Asian type,they had sig-nificantly different effects on the IL-8 secretion levels of AGS cells.Meanwhile,both Cag A ABC and Cag AABCCC which belonged to Western type had significantly different effects on the IL-8 secretion levels too.Conclusion：Cag A virulent factor of H.pylori caused cell hummingbird phenotype and increased the IL-8 secretion lev-els of AGS cells.The number of EPIYA-C motif was positively correlated with hummingbird-cell formation,andthe numbers of EPIYA-C motif as well as EPIYA-D motif were positively correlated with the IL-8 secretion levels of AGS cells.