幽门螺杆菌、奥美拉唑及胃泌素对胃黏膜上皮细胞增生凋亡的影响

Effects of Helicobacter pylori, omeprazole and gastin on the proliferation and apoptosis of gastric epithelial cell

目的 采用细胞实验探讨幽门螺杆菌（Hp）、奥美拉唑与胃泌素之间是否具有相互作用.方法 奥美拉唑、Hp超声粉碎物及胃泌素分别处理不同组细胞后,依次采用MTS和流式羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）染色定量检测胃上皮细胞低分化AGS细胞和永生GES-1细胞增生的变化.另外,采用Hoechst33342染色定性分析细胞形态学的凋亡改变,并且再次运用流式膜联蛋白（Annexin）-Ⅴ/碘化丙啶（PI）定量分析细胞的凋亡.结果 （1）Hp、奥美拉唑处理的胃黏膜细胞AGS和GES-1凋亡比例均高于对照组[Hp及104 nmol/L奥美拉唑处理组凋亡比例分别为（17.20±0.90）%、（12.81±0.78）%比（7.96±1.25）%,（4.60±0.34）%、（6.60±0.76）%比（3.52±0.16）%,均P＜0.05],并且这一作用与奥美拉唑浓度有关.两种细胞增生均明显低于对照组（Hp及104 nmol/L奥美拉唑处理组细胞增生分别为13.35±0.55比37.78±1. 98、47.62±2.40比62.44±4.46,27.15±1.64比32.76±1.57、29.42±1.44比48.86±4.95,均P＜0.05）.（2）经Hp、奥美拉唑及胃泌素两两共同处理后,凋亡方面,除Hp与500 ng/L胃泌素组处理的GES-1细胞凋亡比例较高外[（7.25±0.54）%比（4.60±0.34）%,P＜0.05],其余两种因素处理组差异均无统计学意义.流式检测显示,经Hp与奥美拉唑及胃泌素两两共同处理后,两种细胞增生均低于Hp单个因素处理后（AGS：12.68±0.09、12.28±0.31比13.35±0.55,GES-1：22.06±1.61、18.59±0.09比27.15±1.64,均P＜0.05） 104 nmol/L奥美拉唑和500 ng/L胃泌素共同处理后,两种细胞增生均低于104nmol/L奥美拉唑处理组（33.23±5.98比47.62±2.40、25.97±0.74比29.42±1.44,均P＜0.05）.（3）虽然胃泌素处理后的细胞增生和凋亡改变均不明显,但经奥美拉唑与胃泌素,Hp与胃泌素的组合处理后,两种细胞增生均明显低于对照组（均P＜0.05）.（4）两种细胞改变比较,以AGS的改变更明显.结论 虽然胃泌素对体外胃黏膜上皮细胞没影响,但能显著提高奥美拉唑、Hp对于细胞的作用,说明奥美拉唑、Hp与胃泌素存在相互作用,能共同引起胃黏膜细胞增生减低和调亡增高,促使胃黏膜细胞减少,推测长期质子泵抑制剂治疗与Hp感染可能共同参与了抑酸治疗时萎缩性胃炎的发生.

Objective To explore whether there is an interaction between Helicobacter pylori （H. pylori）, omeprazole and gastrin on the proliferation and apoptosis of gastric epithelial cells. Methods With omeprazole, H. pylori and gastrin as treatment factors, the in vitro changes of gastric epithelial cells AGS and GES-1 were detected by MTS, Hoechst33342 and flow cytometry. Results （1）For a single factor, the gastric mucosal cells （ AGS and GES-1 ） treated by H. pylori or omeprazole show significantly higher apoptosis than that of control group [H. pylori and 104 nmol/L omeprazole group： （ 17.20 ± 0. 90） %,（12. 81 ±0.78）% vs （7.96 ±1.25）% （4.60 ±0.34）%, （6.60 ±0.76）% vs （3.52 ±0. 16）% all P 〈 0. 05]. The gastric mucosal cells treated by H. pylori or omeprazole show significantly lower proliferation than that of control group（ H. pylori and 104 nmol/L omeprazole group： 13. 35 ± 0. 55 vs 37.78 ± 1.98,47.62 ± 2. 40 vs 62.44 ± 4. 46 27. 15 ± 1.64 vs 32. 76 ± 1.57, 29. 42 ± 1.44 vs 48. 86 ± 4. 95 all P 〈0. 05 ）. The differences were statistically significant and the effects were correlated with the concentration of omeprazole. Gastrin had no direct effect on cellular proliferation and apoptosis （ all P 〉 0. 05 ）. （ 2 ） For a combinations of factors, the GES-1 cells treated with H. pylori and 500 ng/L gastrin had a significantly higher ratio [（7.25 ±0. 54） % vs （4. 60 ±0. 34） %, P 〈0. 05], while the other groups just showed a rising trend in the proportion of apoptosis cells, but there was no statistical significance. Flow cytometry analysis showed that combinations of H. pylori, omeprazole and gastrin caused lower proliferation than that of H. pylori group,and all changes were statistically significant （ AGS： 12. 68 ±0. 09, 12. 28 ±0. 31 vs 13. 35 ±0. 55 GES-1：22.06 ± 1.61, 18. 59 ± 0. 09 vs 27.15 ± 1.64 all P 〈 0. 05 ）. And combinations of 104 nmol/L omeprazole and 500 ng/L gastrin caused lower proliferation than that of 104 nmol/L omeprazole group（33. 23 ±5.98 vs 47.62 ± 2. 40 and 25.97 ± 0. 74 vs 29. 42 ± 1.44, both P 〈 0. 05 ）. （3） When comparing the apoptosis and proliferation changes of two cell lines, gastric cancer cells AGS had a more marked changes than immortalized cells GES-1. Conclusion Gastrin significantly enhances the effects of omeprazole and H. pylori on gastric epithelial cells. It is speculated that the factors of omeprazole, H. pylori and gastrin may have a synergistic effect of decreased proliferation and increased apoptosis of gastric mucosal cells. Thus a long-term proton pump inhibitor treatment of H. pylori patients may carry a higher risk of atrophic gastritis. But it needs to be confirmed by further experiments.