营养缺乏对自由生活阿米巴自噬的影响

Effect of Nutritional Stress on Autophagy in Free-Living Amoeba

目的研究在营养缺乏的环境中自由生活阿米巴自噬的变化和自噬结构的形态学特征。方法对照组自由生活阿米巴用涂有大肠埃希菌的琼脂培养基培养。实验组将在大肠埃希菌中培养的阿米巴转移至不含大肠埃希菌的琼脂培养基上培养12 h。扫描电镜下观察不提供细菌的培养环境中阿米巴的形态学变化,透射电镜下观察阿米巴自噬的变化及自噬前体、自噬体和自噬溶酶体的结构特点,图像分析仪测量虫体内自噬结构与细胞质的断面面积。单丹磺酰尸胺(MDC)染色法标记自由生活阿米巴虫体内的自噬体,在共聚焦激光扫描显微镜下观察和定量分析。结果对照组中自由生活阿米巴均为滋养体形式;实验组中,滋养体逐渐向包囊转变。对照组阿米巴虫体内充满细菌碎片,只发生轻微的自噬,自噬结构数目较少。与对照组比较,实验组阿米巴自噬水平显著提高,自噬结构数目增多,自噬前体、自噬体和自噬溶酶体与细胞质的断面面积比增大(P<0.05或0.01);部分阿米巴虫体内残存未消化的细菌碎片。结论在缺乏营养的环境中,自由生活阿米巴由滋养体向包囊转变,虫体内自噬功能显著增强。

Objective To investigate the change of autophagy and morphological characteristics of the autophagic structures in free-living amoeba under nutritional stress.Methods Free-living amoebae were incubated on the agaric solid medium which had been covered with Escherichia coli in control group.In the experiment group,amoebae incubated on the agaric solid medium with E.coli were collected and moved to another solid medium without E.coli and incubated for 12h.The morphological changes of free-living amoeba in the medium without E.coli were viewed with scanning electron microscope.The changes of autophagy and the structural features of the autophagosome precursors,autophagosomes and autophagolysosomes in amoeba were examined with transmission electron microscope,and the cross-section areas of the autophagic structures and cytoplasm were measured with an image analyzer.The autophagosomes in the organism were labeled with monodansylcadaverine （MDC）staining and quantitated using laser scanning confocal microscope.Results In the control group,free-living amoebae were all in the form of trophozoite.In the experiment group,trophozoites were induced to transform to cysts gradually.In control group,amoeba was full of fragment of E.coli.There was merely little autophagy with fewer autophagic structures in amoeba.When compared with the control group,the autophagic abilities of amoeba were enhanced significantly,number of autophagic structures increased in the experiment group.In addition, the ratio of the cross-sectional areas of the autophagic structures to that of the cytoplasm of amoeba was greater （P0.05 or 0.01）.There was fragment of E.coli that was not digested in some of the amoebae.Conclusion In the circumstance of nutritional stress,amoebic trophozoites were induced to transform to cysts gradually.The autophagic ability of free-living amoeba significantly enhanced.