文章摘要
秦〓霞,曹剑秋,柏〓惠,杨〓青,陈 琪.甘氨酸对ATP耗竭诱生性细胞自噬的作用及其机制[J].南京医科大学学报,2009,29(12):1633~1637
甘氨酸对ATP耗竭诱生性细胞自噬的作用及其机制
Effects of glycine on the ATP depletion-induced autophagy in MDCK cells
投稿时间:2009-09-10  
DOI:10.7655
中文关键词: 甘氨酸  自噬  ATP耗竭  AMPK
英文关键词: glycine  autophagy  ATP depletion  AMPK
基金项目:国家自然科学基金面上项目(30370659)
作者单位
秦〓霞 南京医科大学动脉粥样硬化研究中心,江苏 南京〓210029 
曹剑秋  
柏〓惠  
杨〓青  
陈 琪  
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中文摘要:
      目的:研究甘氨酸(Gly)在ATP耗竭性细胞自噬中的作用及其可能机制?方法:用脂质体转染法将绿色荧光蛋白-微管相关蛋白轻链3(GFP-LC3)质粒导入犬肾细胞(MDCK)中,在倒置荧光显微镜下观察细胞内自噬体的生成?同时收集细胞总蛋白,用Western blot检测LC3-Ⅱ/LC3-Ⅰ的表达,并检测AMP激活的蛋白激酶(AMPK)的磷酸化活性改变?结果:MDCK细胞经转染GFP-LC3质粒后,可见细胞浆内散布明显的点状荧光颗粒;转染细胞被剥夺ATP后,发现细胞内自噬体数目明显增多,GFP-LC3-Ⅱ/LC3-Ⅰ比值明显增高,AMPK的磷酸化水平增强?用甘氨酸处理细胞后,发现自噬体较ATP耗竭组明显减少,GFP-LC3-Ⅱ/LC3-Ⅰ比值明显降低,细胞AMPK的磷酸化水平也有所降低?结论:ATP耗竭能诱导细胞的自噬性死亡,Gly可抑制ATP耗竭细胞的自噬,其机制可能与AMPK活性受抑有关?
英文摘要:
      Objective:To investigate the effect of glycine on the ATP depletion-induced autophagy and mechanism underlying it. Methods:GFP-LC3 plasmid was transiently transfected into Madin-Darby canine kidney (MDCK) cells with lipofectamine. Autophagosome in cells was observed under an inverted fluorescence microscope. Expression of GFP-LC3-Ⅱ/LC3-Ⅰ and the phosphorylation activity of AMPK in the transfected cells were analyzed by western blot. Results:We found that the clear punctate fluorescence particles were dispersed in the cytoplasm of the transfected cells. When ATP was deprived off by addition of rotenone and antimycin into MDCK cells,autophagosome and GFP-LC3-Ⅱ/LC3-Ⅰratio were increased and AMPK was activated in cells. Treatment with glycine resulted in decrease in autophagosome and GFP-LC3-Ⅱ/LC3-Ⅰ expression in the ATP-depleted cells. Treatment with glycine also inhibited phosphorylation of AMPK in the ATP-depleted cells. Conclusion:ATP depletion induces an autophagic cell death,which could be inhibited by glycine. The inhibitory effect of glycine on the ATP depletion-induced autophagy may be involved in the depression of AMPK phosphorylation.
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