幽门螺杆菌hp0788基因对GES-1细胞功能影响的初步研究

Preliminary study of how H. pylori gene hp0788 affects the function of GES-1 cells

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,P<0.05);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,P<0.05)。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。

Objectives To create an hp0788-deleted strain （Helicobacter pylori ATCC26695△0788km） and to func- tionally study the role of hp0788 in H. pylori pathogenesis. Methods A knockout plasmid was constructed based on pSJHK. Upstream and downstream sequences （800-1100bp） of the hp0788 gene were amplified as upstream and down- stream homologous arms. These homologous arms were digested with restriction enzymes and ligated into pSJ HK, yield- ing the plasmid pSJHK-0788. This plasmid was transformed into H. pylori ATCC26695 by electroporation to generate an hp0788-deleted strain. The adherence of FITC-labeled the H. pylori to GES-1 was measured using flow cytometry. The viability of GES-1 cells and the rate of GES-1 cell apoptosis were determined in an H. pylori ATCC26695 or H. pylori ATCC26695△0788km and GES-1 co-culturing system. Results The plasmid pSJHK-0788 was transformed into H. pylori ATCC26695 by electroporation to generate an hp0788-deleted strain. FITC-labeled H. pylori ATCC26695 adhered to GES-1 cells at a rate of 100% and H. pylori ATCC26695△0788km adhered to GES-1 cells at a rate of 90.40% （t=2.80, P〈0.05）. After infection with H. pylori ATCC26695 and co-culturing for 8 h, the rate of GES-1 cell apop- tosis increased （15.73± 7.84）% ; after infection and co-culturing for 16 h, the rate of GES-1 cell apoptosis increased （25. 26±5.81） %. After infection with H. pylori ATCC26695△0788km and co-cuhuring for 8 h, the rate of GES-1 cell apoptosis increased （12.46± 12.30）%; after infection and co-culturing for 16 h, the rate of GES-1 cell apoptosis increased （21.13± 10.09） %. After infection with H. pylori ATCC26695 and co-culturing for 8 h, the viability of GES-1 cells decreased 53%; after infection and co-culturing for 16 h, the viability of GES-1 cells decreased 42%. After infection with H. pylori ATCC26695△0788km and co-culturing for 8 h, the viability of GES-1 cells decreased 66% after infection and co-culturing for 16 h, the viability of GES-1 cells decreased 50% （t=3.30, 2.80, -2.93, -2. 76, P〈0. 05）. Coladusion The rate at which H. pylori ATCC26695 adhered to GES-1 cells decreased as a result of the deletion of hp0788 and the apoptosis of GES-1 cells also decreased, but cell viability increased. These results suggested that hp0788 plays an important role in H, pylori pathogenesis.