幽门螺杆菌诱导人胃上皮细胞系(GES-1)发生自噬

Helicobacter pylori induces autophagy of human gastric epithelium cell line(GES-1)

目的观察幽门螺杆菌感染人胃上皮细胞后自噬的发生及变化情况,为进一步研究幽门螺杆菌感染后诱发自噬反应的相关研究奠定基础。方法采用中性红摄入法确定H.pylori感染GES-1细胞的浓度,并以此浓度感染GES-1细胞不同时间。通过Western blot检测感染不同时间点GES-1中LC3I/II或P62蛋白表达,免疫荧光染色观察自噬体的出现。在使用自噬抑制剂3MA、E64d、pepstatin A处理被感染的GES-1后,利用上述方法监测细胞自噬变化情况。结果 H.pylori(22695)感染GES-1细胞最佳浓度为m.o.i=400。在GES-1细胞被感染后2 h即可观察到自噬的发生,且呈时间依赖性,于8 h到达高峰,24 h开始恢复基础水平。使用3MA处理细胞后自噬水平降低,自噬体明显减少。使用E64d和pepstatin A阻断被感染细胞中自噬溶酶体的形成,LC3I/II与P62蛋白因无法通过自噬途径降解而较未处理组增加。结论 H.pylori(22695)能够诱导人胃上皮(GES-1)发生自噬,且能被自噬调节剂调控。

Objective To observe autophagy induced by H.pylori infection and its modulation in human gastric epithelium（GES-1）.Methods GES-1 was infected by H.pylori for different hours with the optimal multiplicity of infection（m.o.i） which was determined by neutral red uptake method（NRU）.Western blot was utilized for detecting the level of LC3-I/II conversion and P62,which indicated the autophagy induction.The formations of autophagosomes were observed via immunofluorescence with microscopy.After the GES-1 infected by H.pylori were treated with 3MA,E64d and pepstatin A,the changes of autophagy were detected by the methods above.Results The optimum m.o.i was 400 for GES-1 infected by H.pylori（22695）.At 2 h p.i,the autophagy induction became detectable.It reached a peak level at 8 h p.i,while it returned to the basic level at 24 h p.i.3MA was able to inhibit autophagy with obviously decrease of autophagosome formation.And E64d and pepstatin A could block autophagy downstream to some extent,which is showed by the aggregation of LC3 and P62.Conclusions H.pylori is capable of inducing autophagy of human gastric epithelium（GES-1）,and the process can be modulated by autophagic regulators.