幽门螺杆菌诱导人胃上皮细胞(GES-1)自噬的研究

 摘要: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感染细胞的最佳浓度，以此浓度分别感染细胞,通过Western Blot检测感染不同时间LC3与p62的表达情况，免疫荧光染色检测自噬体。使用自噬抑制剂3MA、E64d、 pepstatin A干预后，观察感染后自噬变化情况。结果 H. pylori (22695) 感染GES-1细胞最佳浓度为m.o.i=400。感染后2小时即可观察到自噬的发生，且呈时间依赖性，于8小时到达高峰，24小时开始恢复基础水平。使用3MA阻断自噬，自噬体明显减少，且细胞形态欠佳。使用E64d和pepstatin A处理后自噬也受到一定程度的抑制。结论 H. pylori（22695）能够诱导人胃上皮（GES-1）发生自噬，且可被自噬抑制剂所抑制。

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

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.