摘要
【目的】本文研究布鲁氏菌磷酸葡萄糖变位酶(pgm)基因缺失株和PGM蛋白对胚胎滋养层细胞(HPT-8)的损伤作用及其引起炎症反应时细胞因子的变化,探讨布鲁氏菌pgm基因的生物学功能。【方法】本文分别用布鲁氏菌pgm基因缺失株和纯化的PGM蛋白感染胚胎滋养层细胞,通过细胞形态学的观察和酶联免疫反应检测其对细胞的损伤作用以及细胞因子的变化。【结果】本实验获得了纯化的PGM蛋白,成功构建了pgm基因缺失株,采用该基因缺失株免疫小鼠后,采集血液分离血清,虎红平板实验和试管凝集实验结果显示为阴性;pgm缺失株和PGM蛋白感染HPT-8细胞均能诱发贴壁细胞脱落;而且pgm基因缺失株侵袭HPT-8细胞的能力较M5-90明显降低。pgm基因缺失株侵袭HPT-8细胞诱导产生的细胞因子IL-6、TNF-α和LDH均高于M5-90对照组,差异极显著(P<0.01),而细胞因子IL-10的分泌变化不明显,PGM蛋白感染HPT-8细胞时,其细胞因子LDH表达水平高于PBS对照组,差异极显著(P<0.01),而IL-6、IL-10和TNF-α的表达水平明显低于PBS对照组,差异显著(P<0.05)。【结论】本研究表明,PGM蛋白和pgm缺失株可致胚胎滋养层细胞损伤,且引发滋养层细胞细胞因子的表达变化,为进一步研究布鲁氏菌感染宿主细胞的分子机制奠定了基础。
[Objective]We studied the biological function of Brucella phosphoglucomutase（pgm） gene,and detected the changes of human trophoblast cell invaded by the Brucella pgm mutant and PGM protein.[Methods] Human trophoblast cells were infected by the pgm mutant and PGM protein.The changes of cytokines were detected by enzyme-linked immunosorbent assay,and morphology of cells was identified.[Results] PGM protein was purified,and pgm mutant was constructed.The sera of mice immunized by pgm mutant were negative by agglutination test and Standard Tube Agglutination Test for Brucellosis.The cellular morphology of human trophoblast cells infected pgm mutant or PGM protein changed.The adhesion and infection of the pgm mutant reduced more than Brucella vaccine strain M5-90,and human trophoblast cells partially cracked off.The activity of IL-6,TNF-α or lactic dehydrogenae increased in human trophoblast cells infected by the pgm mutant more than Brucella vaccine strain M5-90（P〈0.01）,but not for IL-10.Lactic dehydrogenae in human trophoblast cells infected by the PGM Protein increased more than sodium phosphate buffer（P〈0.01）,whereas IL-6 and TNF-α decreased in human trophoblast cells less than sodium phosphate buffer（P〈0.05）.[Conclusion] The results suggest that the pgm mutant of brucella and PGM protein had the cytotoxic effect for human trophoblast cells with cellular morphology and changes of cytokines.
出处
《微生物学报》
CAS
CSCD
北大核心
2012年第8期1040-1048,共9页
Acta Microbiologica Sinica
基金
国家"973项目"(2010CB30203)
国家自然科学基金(31001046/30960288)
高层次人才科研启动资金(RCZX200914)~~
