幽门螺杆菌对胃黏膜细胞DNA依赖蛋白激酶催化亚单位和Ku70/Ku80二聚体蛋白体内外表达的影响

Effects of Helicobacter pylori on the expression of catalytic subunit of the DNA-dependent protein kinase and Ku70/Ku80 heterodimer in gastric mucosa epithelial cells in vivo and in vitro

目的 探讨H.pylori对胃黏膜细胞DNA依赖蛋白激酶催化亚单位（DNA-PKcs）和Ku70/Ku80二聚体在体内外表达的影响.方法 应用Western印迹法检测H.pylori作用后1、3、6、12和24 h的胃黏膜上皮细胞（GES-1）和胃腺癌细胞（AGS）中DNA-PKcs和Ku70/Ku80二聚体的蛋白表达情况.采用H.pylori灌胃蒙古沙鼠,于感染第6和12个月处死动物,取胃黏膜组织行免疫组织化学染色,检测DNA-PKcs和Ku70/Ku80二聚体的蛋白表达情况.采用t检验和卡方检验进行统计学分析.结果 H.pylori作用后1h,DNA-PKcs蛋白在GES-1中的相对表达量为1.16±0.09,高于未感染H.pylori组的1.04±0.31,差异有统计学意义（t=4.67,P＜0.05）.H.pylori作用后1、3、6、12和24 h,Ku70/Ku80二聚体蛋白在GES-1中的相对表达量分别为1.58±0.32、1.84±0.40、1.97±0.35、3.72±1.42和3.74±1.56,均高于未感染H.pylori组的1.24±0.31,差异均有统计学意义（t=3.57、4.20、5.03、8.11、8.14,P均＜0.05）；Ku70/Ku80二聚体蛋白在AGS中的相对表达量分别为4.69±0.87、3.67±0.67、2.41±0.24、1.35±0.35和1.32±0.10,均低于未感染H.pylori组的4.84±0.76,差异均有统计学意义（t=34.13、27.68、19.81、4.47、5.69,P均＜0.05）.蒙古沙鼠模型中,DNA PKcs蛋白在未感染H.pylori组不表达（0/25）,在感染H.pylori组中的总阳性率为98.1％（53/54）,两组差异有统计学意义（x2=74.55,P＜0.01）；Ku70/Ku80二聚体蛋白在未感染H.pylori组中的总阳性率为92.0％（23/25）,在感染H.pylori组中的总阳性率为68.5％（37/54）,两组差异有统计学意义（x2=5.16,P＜0.05）.结论 H.pylori感染在体内外能通过改变胃黏膜DNA PKcs和Ku70/Ku80二聚体蛋白的表达水平,影响细胞DNA损伤修复,导致胃黏膜病变.

Objective To explore the effects of Helicobacter pylori （H.pylori） on the expression of catalytic subunit of the DNA-dependent protein kinase （DNA-PKcs） and Ku70/Ku80 heterodimer in gastric mucosa epithelial cells in vivo and in vitro.Methods After treated with H.pylori for one,three,six,12 and 24 hours,the expressions of DNA-PKcs and Ku70/Ku80 heterodimer in gastric epithelial cells （GES） 1 and gastric adenocarinoma cells （AGS） were detected by Western blot.Mongolian gerbils were gavaged with H.pylori,and were sacrificed after infected for six and 12 months.The gastric mucosa tissues were taken for immunohistochemistry to detect the expressions of DNA-PKcs and Ku70/Ku80 heterodimer at protein level.The data were analyzed by t test and chi-square test.Results After H.pylori infection for one hour,the relative quantity of the expression of DNA-PKcs in GES-1 was 1.16±0.09,which was higher than that of non infected group （1.04±0.31） and the difference was statistically significant （t=4.67,P〈0.05）.After infected by H.pylori for one,three,six,12 and 24 hours,the relative quantities of the expressions of Ku70/Ku80 heterodimer in GES-1 were 1.58±0.32,1.84±0.40,1.97±0.35,3.72±1.42 and 3.74±1.56,respectively,all were higher than that of non infected group （1.24±0.31） and the differences were statistically significant （t=3.57,4.20,5.03,8.11 and 8.14,all P〈0.05）.The relative quantities of the expressions of Ku70/Ku80 heterodimer in AGS were 4.69 ± 0.87,3.67 ± 0.67,2.41±0.24,1.35±0.35 and 1.32±0.10 after H.pylori infected for one,three,six,12 and 24 hours,respectively,all were lower than that of no H.pylori infected group （4.84 ± 0.76） and the differences were statistically significant （t=34.13,27.68,19.81,4.47 and 5.69,all P〈0.05）.In Mongolian gerbil models,DNA-PKcs did not express in H.pylori negative group （0/25）,the total positive rate of H.pylori infected group was 98.1% （53/54）,the difference between the two groups was statistically significant （x2 =74.55,P〈0.01）.The total positive rate of Ku70/Ku80 heterodimer in H.pylori negative group was 92.0% （23/25） and in H.pylori infected group was 68.5% （37/54）,the difference between the two groups was statistically significant （x2=5.16,P〈0.05）.Conclusion H.pylori infection affected cellular DNA damage repair through changing the expression of DNA-PKcs and Ku70/Ku80 heterodimer in gastric mucosa in vivo and in vitro,which may cause gastric mucosal lesions.