幽门螺杆菌粘附素HpaA诱导T细胞高表达IL-21促进AGS细胞迁徙的研究

IL-21 secretion by adhesin HpaA-stimulated T cells from Helicobacter pylori improves AGS cell migration

目的验证幽门螺杆菌(Helicobacter pylori,Up)粘附素HpaA通过诱导T细胞分泌IL-21加剧其感染后促进胃粘膜损伤及胃癌转移的作用。方法收集56例确诊Hp感染患者临床内窥镜取得的胃粘膜,以及经根治后再次通过内窥镜取得胃粘膜,通过RT-PCR和Western blot比较其IL-21、MMP-2、MMP-9及其基因在Hp感染前后胃粘膜中的表达水平,并克隆构达、纯化粘附素HpaA。取健康未感染成年人外周血PBMC,磁珠分选获得CD3+T细胞。使用重组HpaA刺激分选获得的细胞,采用ELISA试剂盒检测其上清中IL-21的水平。培养胃粘膜AGS细胞系并使用不同手段干预培养环境中的IL-21水平,采用Western blot检测AGS细胞MMP-2及MMP-9的表达。并通过Transwell实验验证IL-21对AGS细胞迁徙的影响。结果Western blot检测Hp感染患者的胃粘膜中IL-21.MMP-2和MMP-9表达水平显著高于根治后的胃粘膜(PV0.05)。成功构建PET-28a/HpaA重组质粒,表达并纯化的粘附素HpaA能诱导CD3+T细胞分泌高水平的IL-21。AGS细胞试验显示-IL-21诱导AGS细胞MMP-2及MMP-9的表达水平显著升高;使用抗体阻断IL-21以及使用适配子HA6(之前报道的能够特异性结合HpaA的配基)阻断HpaA后MMP-2及MMP-9的表达水平显著下降,且AGS细胞迁徙水平降低。结论粘附素HpaA通过诱导T细胞产生IL-21,继而通过MMP对促进胃粘膜损伤及胃癌转移有重要意义。

Objective To dermonstrate that adhesin HpaA of Helicobacter pylori can intensify mucosal injury and gas-tric cancer metastasis by IL-21 from induced T-cells.Methods Gastric mucosal biopsy specimens were collected while 56 patients seen at the Second Peoples Hospital of Wubu were infected with H.pylori and after eradication of H,pylori.Levels of IL-21,MMP-2,and MMP-9 from biopsy specimens were detected using RT-PCR and Western botting.Re-combinant adhesin HpaA was cloned,expressed,and purified.Magnetic cell sorting was performed,CD3+T cells were isolated from PBMCs from healthy donors,and the level of IL-21 in supernatant fluid was detected using ELISA.AGS cells were cultured,and MMP-2 and MMP-9 were detected in AGS cells with W estern blotting after different interven-tions.The IL-21-induced migration of AGS cells was detected with a Transwell invasion assay.Results Western blot-ting indicated that a higher level of IL-21 MMP-2 and MMP-9 was expressed in biopsy specimens while patients were in-fected with H.pylori than after H.pylori was eradicated.Recombinant HpaA was obtained to study its function.Data indicated that HpaA increased IL-21 secretion by inducing the proliferation of CD3+T cells in vitro.MMP-2 and MMP-9 were expressed in AGS cells stimulated with IL-21.Use of antibodies to block IL-21 activity and aptamer HA6(a ligand specifically binding to HpaA reported previously)decreased the level of MMP-2 and MMP-9 and the migration of AGS cells.Conclusion Results suggested that adhesin HpaA plays a significant role via IL-21 from induced T-cells during gastric mucosa injury and gastric cancer metastasis caused by infection with H.pylori.