幽门螺杆菌hp0788基因对GES-1细胞功能影响的初步研究

发布时间：2018-03-21 08:19

本文选题：幽门螺杆菌　切入点：基因敲除　出处：《中国病原生物学杂志》2017年07期 　论文类型：期刊论文

【摘要】：目的构建幽门螺杆菌ATCC26695hp0788基因敲除突变菌株(ATCC26695△0788km),观察hp0788基因对GES-1细胞功能的影响。方法以基因敲除质粒载体pSJHK构建基因敲除质粒,在hp0788基因的上下游分别扩增800~1100bp的基因片段作为同源臂,经限制性内切酶酶切后与质粒载体连接构建基因敲除质粒pSJHK-0788,通过电击转化构建hp0788基因敲除突变菌株,以FITC标记法检测其对GES-1细胞黏附能力的影响;以感染复数(MOI)为200:1构建幽门螺杆菌与GES-1细胞共培养体系,比较ATCC26695和ATCC26695△0788km对GES-1细胞凋亡及活性的影响。结果构建了hp0788基因双交换敲除质粒,电击转化获得hp0788基因敲除突变菌株。FITC标记ATCC26695和幽门螺杆菌ATCC26695△0788km与GES-1细胞混匀后采用流式细胞术检测FITC的荧光强度,ATCC26695组的黏附率记为100%,ATCC26695△0788km组黏附率为90.40%,差异均有统计学意义(t=2.80,P0.05);ATCC26695与ATCC26695△0788km分别与GES-1细胞共培养8h和16h,流式细胞术检测细胞凋亡率分别为(15.73±7.84)%、(25.26±5.81)%和(12.46±12.30)%、(21.13±10.09)%,细胞增殖-毒性检测试剂盒检测细胞活力分别为53%、40%和66%、50%,差异均有统计学意义(t值分别为3.30,2.80,-2.93,-2.76,P0.05)。结论hp0788基因敲除幽门螺杆菌ATCC26695对GES-1细胞的黏附率下降,并使GES-1凋亡率下降,而细胞活力增高。表明hp0788基因是影响幽门螺杆菌ATCC26695感染GES-1细胞后引起细胞凋亡和活性变化的重要基因。
[Abstract]:Objective to construct a mutant strain of Helicobacter pylori ATCC26695hp0788 gene knockout strain ATCC26695 0788kmand to observe the effect of hp0788 gene on the function of GES-1 cells. Methods the gene knockout plasmid vector pSJHK was used to construct the gene knockout plasmid. In the upstream and downstream of the hp0788 gene, 800 ~ 1100bp gene fragment was amplified as the homologous arm. After restriction endonuclease digestion, the plasmid pSJHK-0788 was ligated with plasmid vector to construct the mutant pSJHK-0788. The mutant strain of hp0788 gene knockout was constructed by electroporation. FITC labeling method was used to detect its effect on the adhesion of GES-1 cells, and the co-culture system of Helicobacter pylori and GES-1 cells was constructed with infected plural moi as 200: 1. The effects of ATCC26695 and ATCC26695 0788km on the apoptosis and activity of GES-1 cells were compared. Results the double exchange knockout plasmid of hp0788 gene was constructed. Electroporation of hp0788 gene knockout mutant strain. FITC-labeled ATCC26695 and Helicobacter pylori ATCC26695 0788km mixed with GES-1 cells. The fluorescence intensity of FITC was detected by flow cytometry. The adhesion rate of ATCC26695 group was recorded to be 90.40 in ATCC26695 0788km group. There were significant differences between ATCC26695 and ATCC26695 0788km in cultured GES-1 cells for 8 hours and 16 hours, respectively. The apoptotic rates were 25.26 卤5.81% and 12.46 卤12.30% by flow cytometry, respectively. The activity of cells detected by proliferation-toxicity test kit was 5340% and 6650%, respectively. The t value of hp0788 gene knockout of Helicobacter pylori ATCC26695 is 3.30 ~ 2.80 ~ 2.93- 2.76-P0.05. Conclusion the adhesion rate of hp0788 knockout helicobacter pylori ATCC26695 to GES-1 cells is decreased. The apoptosis rate of GES-1 was decreased and the cell viability was increased, which indicated that hp0788 gene was an important gene affecting the apoptosis and activity of Helicobacter pylori ATCC26695 infected GES-1 cells.
【作者单位】：滨州医学院病原生物学教研室;
【基金】：国家自然科学基金项目(No.81471561) 山东省高等学校科技计划项目(No.J15LK02) 滨州医学院科研启动基金项目(No.BY2014KYQD10,BY2015KYQD09)
【分类号】：R378

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