幽门螺杆菌空泡毒素活性的体外观察

Observations onvacuolating toxin of Helicobacter pylori in vitro

目的研究幽门螺杆菌(Hp)液体浓缩上清液对胃上皮细胞的空泡毒性作用,为进一步开展空泡变性细胞毒素(VacA)的纯化及探讨VacA的致病机制奠定基础。方法将Hp菌株液体培养上清液制成粗制VacA蛋白,粗制VacA经倍比稀释(1∶160～1∶2)后与胃癌SGC7901细胞共同孵育后观察胃癌细胞形态变化,同时观察1∶2及1∶5滴度组于0、2、4、8、16和24h时的空泡活性。采用中性红摄入法(NRU)评价空泡活性。结果1∶2及1∶5滴度组胃癌细胞于24h时空泡形成细胞达100%,空泡细胞比例随毒素滴度递减。孵育24h时粗制VacA产生空泡活性的最小剂量约为6μg,超过120μg可导致细胞死亡。当毒素滴度为1∶2时,孵育4h即产生明显空泡[550nm光吸收值(A550)=0.43±0.06],至16h达最高吸收值(A550=0.71±0.03),至24h时因细胞大量死亡,A550反而下降至0.06±0.04,与空白对照组的差异无显著性(P>0.05)。1∶5毒素滴度的时间曲线较1∶2滴度更恰当反映出空泡活性随孵育时间变化的特性。结论Hp液体培养上清浓缩液对胃癌细胞具有明显的空泡毒性作用,VacA导致的空泡毒性具有明显的浓度及时间依赖性,且易受多种因素的影响,适当的浓度及孵育时间是VacA致病机制研究过程中的关键因素。

Objective To investigate the toxic effect of vacuolating toxin on gastric epithelial cells in Helicobacter pylori supernatant concentrate. Methods The concentrated supernatant of Helicobacter pylori was used as VacA toxin. Serially diluted VacA （1 ： 160-1 ： 2） were incubated with SGC-7901 cells for 24 h. The vacuolating toxic effect at different times of 0, 2, 4, 8, 16 and 24 hours in group 1 ： 2 and 1 ： 5 were also observed, which were evaluated by neutral red uptake （NRU） assay. Results Percentage of vacuolated cells reached 100K at 24 h in 1 ： 5 and 1 ： 2 dilution and decreased with the concentration of rough VacA. Vacuolating activity also changed with time. The vacuoles could be seen after 4 h at dilution 1 ： 2（A550：0. 43±0.06）, and reached the maximum at 16 h（A550 =0.71±0.03）. A550 abnormally decreased at 24 h due to the death of cells （0.06±0.04, P〉0.05 vs control）. The dilution curve of 1 ： 5 reflected the vacuolating activity more correctly than that of dilution 1 ： 2. Conclusion Hellcobacter pylori supernatant concentrate has obviously vacuolating toxicity on gastric cancer cells, which depends on incubating time and concentration of VacA. Optimized incubating time and dilu- tion are the key factors in the protocol of VacA study. （Shanghai Med J, 2005,28：858-861）