幽门螺旋杆菌提取液体外诱导胃上皮细胞线粒体DNA变异的研究

Mitochondrial DNA mutations in gastric endothelial cells induced by extract of helicobacter pylori in vitro

目的 探讨幽门螺旋杆菌（helicobacter pylori,HP）感染与线粒体DNA（mitochondrial DNA,mtDNA）碱基突变之间的关系.方法 HP11638（CagA＋,VacA＋）和HP11638突变株（HP11638M,CagA＋,VacA-）的提取液与人胃腺癌上皮细胞（human gastric adenocarcinoma epithelial cell,AGS）共同孵育后,收集细胞.提取mtDNA,PCR扩增mtDNA Cox-Ⅰ、Cox-Ⅱ、Cox-Ⅲ、ATPase6、ATPase8、Cytb基因和D-Loop区后测序.结果 AGS细胞mtDNA突变率与两株HP提取液呈浓度及时间依赖关系,经HP11638提取液刺激的AGS细胞mtDNA突变率高于经HP11638M提取液的刺激.在所有616个mtDNA D-Loop区的突变中,489个为点突变,81个为插入,46个为缺失.70.9%（437/616）的突变属于GC→AT和AT→GC的转换.85%（17/20）受刺激的AGS细胞的mtDNA D-Loop区的303PolyC、16184PolyC和514CA等位点发生突变.Cox-Ⅰ、Cox-Ⅱ、Cox-Ⅲ、ATPase6和ATPase8基因未发现突变位点,Cytb基因序列发现3个异质性突变.结论 HP能引起mtDNA特别是D-Loop区的突变积累,VacA蛋白参与了这一过程.

Objective To investigate the relationship between the helicobacter pylori （HP） infection and the genetic instability of mitochondrial DNA （mtDNA） in human gastric adenocarcinoma epithelial cells （AGS）. Methods After treated with extracts of HP11638 （CagA＋,VacA＋） or Hp11638 mutant strain （CagA＋ ,VacA-）, AGS cells were collected, and mitochondrial DNA was extracted and Cox- Ⅰ , Cox-Ⅱ,Cox-Ⅲ, ATPase6, ATPase8 and Cytb genes and the D-Loop region were amplified by PCR and then sequenced. Results The mutation rates of the mtDNA in AGS cells were correlated with the extracts of the two HP strains in a concentration- and time-dependent manner. But the mtDNA mutation rate in AGS cells treated with the HP11638 extract was higher than that treated with the Hp11638 mutant extract. Total of 616 mutations in D-Loop region were detected, including 489 point mutations, 81 insertions and 46deletions. Among them, 70. 9% （437/616） belonged to GC to AT and AT to GC transition. Seventeen out of 20 （85%） AGS cells treated with extract of HP had mutations in 303PolyC, 16184PolyC and 514CAregions of mtDNA D-Loop. No mutation was detected in Cox-Ⅰ, Cox-Ⅱ , Cox-Ⅲ, ATPase6 and ATPase8genes, three point mutations were found in the Cytb gene. Conclusion HP can cause the accumulation of mutations in mtDNA, in particular, in the D-Loop region, and the VacA participated in the process.