幽门螺杆菌VacA通过激活NF-κB诱导巨噬细胞分泌及凋亡

发布时间：2018-06-28 07:54

本文选题：幽门螺杆菌 + 空泡毒素　；参考：《南华大学》2008年硕士论文

【摘要】： 目的:构建pDsRed-Monomer-C1/vacA N端真核表达载体,研究幽门螺杆菌(Helicobacter pylori, H. pylori)空泡毒素单一毒力决定簇对THP-1巨噬细胞分泌和凋亡的影响,以及核因子kappa B (nuclear factor kappa B,NF-κB)在调节VacA诱导的THP-1巨噬细胞功能中的作用。方法:用PRIMER5.0软件设计引物,PCR扩增vacA全基因;将PCR产物纯化后插入真核表达载体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鉴定VacA蛋白在细胞中的表达;电子显微镜和中性红摄取试验检测巨噬细胞的空泡样变;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NO水平;荧光探针DCFH-DA检测培养上清的ROS;流式细胞仪检测细胞的凋亡率;凝胶阻滞试验(electrophoretic mobility gel shift assay ,EMSA)检测NF-κB的核转位。结果: (1)双酶切及测序结果显示,成功构建真核表达载体pDsRed-Monomer-C1/vacA; (2)转染后24h用倒置荧光显微镜观察发现,空质粒组表达的单纯红色荧光蛋白,其荧光在细胞内呈弥散性分布,而重组质粒组表达的VacA与红色荧光蛋白的融合蛋白,在细胞质形成聚集的荧光颗粒;Western-blot结果发现重组质粒转染组出现一条免疫反应带,而空质粒转染组和THP-1巨噬细胞组未见免疫反应带; (3)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;中性红摄取实验显示转染后6h,重组质粒组中性红摄取量明显升高,与对照组相比差异有显著性(P 0.05);而转染后24h,重组质粒组的吸光度达到最高; (4)转染后6h,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(P0.05);且分别于转染后6h或24h到达峰值; (5)转染后6h或12h,重组质粒组培养上清中NO、ROS含量明显高于阴性对照组(P0.05);且于转染后24h达到高峰; (6)转染后16h,重组质粒组的凋亡率明显升高,与阴性对照组相比,差异有显著性(P㩳0.05); (7)重组质粒组加NF-κB的特异性抑制剂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显著性(P 0.05); (8) EMSA实验显示,转染后3h细胞核内有活化的NF-κB,且于转染后12h活性最强。结论: (1)成功构建了pDsRed-Monomer-C1/vacA N端真核表达载体; (2) 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; (3) VacA蛋白瞬时高表达诱导THP-1巨噬细胞凋亡; (4) NF-κB可能参与调节VacA诱导的THP-1巨噬细胞的分泌和凋亡。
[Abstract]:Aim: to construct pDsRed-Monomer-C1 / Vaca N-terminal eukaryotic expression vector and to study the effect of single virulence determinant of Helicobacter pylori (H. pylori) vacuole toxin on the secretion and apoptosis of THP-1 macrophages. And the role of nuclear factor kappa B (nuclear factor kappa BnNF- 魏 B in regulating the function of THP-1 macrophages induced by Vaca. Methods: the whole vacA gene was amplified by primer 5.0 primer, purified and inserted into eukaryotic expression vector pDsRed-Monomer-C1. The expression of Vaca protein in THP-1 macrophages was detected by Western-blot after restriction endonuclease digestion and sequencing. Electron microscopy and neutral red uptake assay were used to detect the level of no in the supernatant of macrophages. Fluorescence probe DCFH-DA was used to detect the ros of culture supernatant; flow cytometry was used to detect the apoptosis rate of cells; and gel block assay (electrophoretic mobility gel shift assay) was used to detect the nuclear translocation of NF- 魏 B. Results: (1) double enzyme digestion and sequencing showed that the eukaryotic expression vector pDsRed-Monomer-C1 / vacAwas successfully constructed. The fluorescence of Vaca and red fluorescent protein expressed in the recombinant plasmid group was diffusely distributed in the cells. The results of Western-blot analysis showed that there was an immunoreactive band in the recombinant plasmid transfection group, and the fusion protein between Vaca and red fluorescent protein was expressed in the recombinant plasmid group, and the results of Western-blot analysis showed that there was an immunoreactive band in the transfected group. However, no immunoreactive bands were found in the empty plasmid transfection group and THP-1 macrophage group. (3) when the recombinant plasmid was transfected into THP-1 macrophage for 24 h, vacuoles of varying size were found in some cells. Neutral red uptake test showed that the uptake of neutral red increased significantly in the recombinant plasmid group at 6 h after transfection. The absorbance of the recombinant plasmid group reached the highest at 24 h after transfection, (4) at 6 h after transfection, the content of TNF- 伪 and IL-1 尾 in the supernatant of the recombinant plasmid group was significantly higher than that in the negative control group (P 0.05), and the content of TNF- 伪 and IL-1 尾 in the culture supernatant of the recombinant plasmid group was significantly higher than that in the negative control group (P0.05). (5) at 6 h or 12 h after transfection, the NON-Ros content in the supernatant of the recombinant plasmid group was significantly higher than that in the negative control group (P0.05), and reached the peak at 24 h after transfection; (6) at 16 h after transfection, NON-Ros content in the culture supernatant of the recombinant plasmid group was significantly higher than that in the negative control group (P0.05). The apoptosis rate of the recombinant plasmid group was significantly higher than that of the control group. Compared with the negative control group, there was a significant difference in the secretion of IL-1 尾 -TNF- 伪 and the apoptosis rate of the cells in the recombinant plasmid group (P0. 05); (7) and the specific inhibitor of NF- 魏 B (PDTC). Compared with the recombinant plasmid group, there was a significant difference (P0. 05); (8) EMSA assay showed that there was activated NF- 魏 B in the nucleus 3 h after transfection, and the activity of NF- 魏 B was the highest at 12 h after transfection. Conclusion: (1) pDsRed-Monomer-C1 / Vaca N-terminal eukaryotic expression vector was successfully constructed; (2) Vaca protein transient overexpression up-regulated the secretion of IL-1 尾 -TNF- 伪 no and ROSby THP-1 macrophages; (3) transient overexpression of VacA protein induced THP-1 expression. (4) NF- 魏 B may be involved in regulating the secretion and apoptosis of THP-1 macrophages induced by VacA.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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