幽门螺杆菌VacA N端在巨噬细胞中表达对其细胞因子 分泌功能的影响

Helicobacter pylori VacA up-regulates secretion of macrophages by Transfec-tion of vacA eukaryotic expression vector

摘要:

构建pDsRed-Monomer-C1/vacA N端真核表达载体, 研究幽门螺杆菌空泡毒素单一毒力决定簇对THP-1巨噬细胞细胞因子分泌的影响。PCR扩增vacA目的基因片段, 克隆入真核表达载体pDsRed-Monomer-C1中, 经酶切、PCR及测序鉴定后, 转染 THP-1巨噬细胞中, Western blot和荧光显微镜鉴定VacA蛋白在细胞中的表达; 电子显微镜和中性红摄入法观察巨噬细胞的空泡样变; ELISA法检测巨噬细胞培养上清TNF-a、IL- 1b含量。重组质粒转染THP-1巨噬细胞24h, 部分细胞胞浆中出现大小不等的空泡, 且重组质粒组培养上清中TNF-a、IL-1b含量明显高于空质粒组和阴性对照组(P<0.001), 二硫代氨基甲酸吡咯烷(pyrrolidine dithiocarbamate, PDTC)下调细胞因子的分泌。结果显示, 成功构建pDsRed-Monomer-C1/vacA真核表达载体; VacA蛋白瞬时高表达上调THP-1巨噬细胞分泌TNF-a、IL-1b; 核因子kB(nuclear factor kappaB, NF-kB)可能参与调节VacA诱导的THP-1巨噬细胞的分泌。

关键词:幽门螺杆菌; 空泡毒素; 真核表达载体; 巨噬细胞; 细胞因子

Abstract:

We constructed a recombinant plasmid containing the N-terminus gene of vacA gene of Helicobacter pylori and studied the effect of VacA on the secretion of macrophages as an individual virulence determinant. VacA gene amplified by Polymerase Chain Reaction (PCR) from Helicobacter pylori was cloned into eukaryotic expression vector pDsRed-Monomer-C1. The recombinant plasmids were verified by restriction endonucleases analysis and nucleotide se-quencing. Then the recombinant plasmids pDsRed-Monomer-C1/vacA were transfected into macrophages. Their expres-sion in macrophages was examined by Western blot and fluorescence microscope. Vacuolated phenotype in macrophages was observed by electron microscopy and neutral red uptake. The cytokine content of TNF-α or IL-1β in the culture me-dium was tested quantitatively with Enzyme Linked Immunosorbent Assay( ELISA) kit, respectively. The effect of pyr-rolidine dithiocarbamate (PDTC), an inhibitor of NF-kB, on the secretion of macrophages transfected with the recombi-nant plasmids, was also studied. Restriction endonucleases analysis and nucleotide sequencing showed that the eukaryotic expression recombinant pDsRed-Monomer-C1/vacA was successfully constructed. A clear vacuolated phenotype devel-oped in some of macrophages transfected with the recombinant plasmids. VacA over-expressed increased the level of TNF-a and IL-1b. PDTC decreased the production of TNF-a and IL-1b induced by VacA. In conclusion, we have suc-cessfully constructed the eukaryotic expression plasmid encoding VacA. The over-expression of VacA fusion protein can up-regulate secretion of macrophages. Activation of NF-kB is probably involved in VacA induced cytokines production.

Key words:Helicobacter pylori; VacA; eukaryotic expression vector; Macrophages; Cytokine