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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。
AIM: To investigate the role of glycine in autophagy of ATP depletion cells and its possible mechanism. METHODS: GFP-LC3 plasmid was transfected into canine kidney cells (MDCK) by lipofection. The formation of autophagosomes was observed under an inverted fluorescence microscope. Meanwhile, the total cellular protein was collected. The expression of LC3-Ⅱ / LC3-Ⅰ was detected by Western blot and the phosphorylation activity of AMP-activated protein kinase (AMPK) was detected. Results: GFP-LC3 plasmid transfected MDCK cells showed obvious dot-like fluorescent particles dispersed in the cytoplasm. After being deprived of ATP, the number of autophagosomes was significantly increased in GFP-LC3-Ⅱ / LC3 -Ⅰ ratio increased significantly, AMPK phosphorylation increased. After treated with glycine, the number of autophagosomes was significantly decreased compared with ATP depletion group, the ratio of GFP-LC3-Ⅱ / LC3-Ⅰ was significantly decreased, and the phosphorylation of AMPK was also decreased. CONCLUSION: ATP depletion can induce autophagic cell death. Gly inhibits autophagy in ATP-depleted cells, which may be related to inhibition of AMPK activity.