CAJ | 학술논문

目的研究幽门螺杆菌(Helicobacterpylori)空泡毒素(VacA)单一毒力决定簇对THP-1巨噬细胞分泌和凋亡的影响,以及核因子KB(nuclear factor KB,NF-KB)在调节VacA诱导的THP-1巨噬细胞功能中的作用.方法将pDsRed-Monomer-Cl/vacA转染THP-1巨噬细胞,ELISA法检测巨噬细胞培养上清IL-1β、TNF-α含量;Griess试剂分析培养上清的一氧化氮(NO)水平;荧光探针DCFH-DA检测培养上清的活性氧(ROS);流式细胞仪检测细胞的凋亡率;凝胶阻滞试验(electro-phoretic mobility gel shift assay,EMSA)检测NF-kB的核转位.结果重组质粒转染后6 h,重组质粒组培养上清中TNF-α、IL-1β含量明显高于阴性对照组(P<0.05);且分别于转染后6 h或24 h到达峰值;转染后6 h或12 h,重组质粒组培养上清中NO、ROS含量明显高于阴性对照组(P<0.05),且于转染后24 h达到高峰.转染后16 h,重组质粒组的凋亡率明显升高,与阴性对照组相比,差异有统计学意义(P<0.05).重组质粒组加NF-kB的特异性抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)后,IL-1β、TNF-α、NO、ROS的分泌量和细胞的凋亡率明显降低,与重组质粒组相比差异有统计学意义(P<0.05).EMSA试验显示,转染后3h细胞核内有活化的NF-kB,且于转染后12h活性最强.结论 VacA蛋白瞬时高表达上调THP-1巨噬细胞分泌IL-1β、TNF-α、NO和ROS;VacA蛋白瞬时高表达诱导THP-1巨噬细胞凋亡;NF-kB可能参与调节VacA诱导的THP-1巨噬细胞的分泌和凋亡.
목적 연구유문라간균(Helicobacterpylori)공포독소(VacA)단일독력결정족대THP-1거서세포분비화조망적영향,이급핵인자KB(nuclear factor KB,NF-KB)재조절VacA유도적THP-1거서세포공능중적작용.방법 장pDsRed-Monomer-Cl/vacA전염THP-1거서세포,ELISA법검측거서세포배양상청IL-1β、TNF-α함량;Griess시제분석배양상청적일양화담(NO)수평;형광탐침DCFH-DA검측배양상청적활성양(ROS);류식세포의검측세포적조망솔;응효조체시험(electro-phoretic mobility gel shift assay,EMSA)검측NF-kB적핵전위.결과 중조질립전염후6 h,중조질립조배양상청중TNF-α、IL-1β함량명현고우음성대조조(P<0.05);차분별우전염후6 h혹24 h도체봉치;전염후6 h혹12 h,중조질립조배양상청중NO、ROS함량명현고우음성대조조(P<0.05),차우전염후24 h체도고봉.전염후16 h,중조질립조적조망솔명현승고,여음성대조조상비,차이유통계학의의(P<0.05).중조질립조가NF-kB적특이성억제제필각완이류대안기갑산염(PDTC)후,IL-1β、TNF-α、NO、ROS적분비량화세포적조망솔명현강저,여중조질립조상비차이유통계학의의(P<0.05).EMSA시험현시,전염후3h세포핵내유활화적NF-kB,차우전염후12h활성최강.결론 VacA단백순시고표체상조THP-1거서세포분비IL-1β、TNF-α、NO화ROS;VacA단백순시고표체유도THP-1거서세포조망;NF-kB가능삼여조절VacA유도적THP-1거서세포적분비화조망.
Objective To study the effect of VacA on the secretion of THP-1 macrophages as an individual virulence determinant, and the effect of NF-kB on the secretion of THP-1 macrophages. Methods The recombinant plasmid pDsRed-Monomer-Cl/vacA was transfected into macrophages. The cytokine con-tent of TNF-α or IL-1β in the culture medium was tested quantitatively with ELISA kit, respectively. The content of NO or ROS in the culture medium was tested with Griess reagent or DCFH-DA fluorescent probe. The apoptosis rate of macrophages was tested by flow cytometry. The effect of PDTC, an inhibitor of NF-kB, on the secretion and apoptosis of macrophages transfected with the recombinant plasmids, was also studied. The activity of NF-kB was examined in THP-1 cells by electrophoretic mobility gel shift assay(EMSA). Re-suits At 6 h after transfection, the level of TNF-α and IL-1 β in macrophages transfected with the recombi-nant plasmids was significantly higher than that of the control group (P <0.05). At 6 h or 12 h after trans-fection, the level of NO and ROS in macrophages transfected with the recombinant plasmids was significantly higher than that of the control group (P <0.05). At 16 h after transfection, the apoptosis rate of macropha-ges transfected with the recombinant plasmids was significantly higher than that of the control group (P < 0.05). PDTC decreased the production of TNF-α, IL-1 β, NO, ROS and apoptosis rate induced by VacA. VacA was found to trigger NF-kB activation. Conclusion The over-expression of VacA fusion protein can up-regulate secretion and apoptosis of macrophages. Activation of NF-kB is probably involved in the produc-tion of TNF-α, IL-1β, NO, ROS and apoptosis induced by VacA.