欢迎访问《解剖学报》官方网站！今天是
English
酒精诱导嗜铬细胞瘤细胞自噬及其与P62作用的关系
Effects of ethanol on autophagy and the role of P62 in ethanol-induced autophagy in pheochromocytoma cells
目的 采用不同浓度酒精作用于大鼠嗜铬细胞瘤（PC12）细胞，观察细胞自噬发生及其与P62变化的关系。 方法 用四甲基偶氮唑盐比色法（MTT）观察酒精对PC12细胞生存率的影响；间接免疫荧光法检测细胞自噬标志性蛋白LC3和P62的变化；高内涵活细胞成像系统检测细胞LC3荧光强度；透射电镜检测细胞自噬的超微结构；Western blotting方法检测P62蛋白量的表达。 结果 50～800mmol/L浓度酒精对PC12细胞的增殖有显著的抑制作用，呈浓度依赖性。酒精致PC12细胞自噬标志性蛋白LC3在细胞核周围密度增高，并与P62形成点状聚集共定位，其中在200mmol/L浓度酒精作用2h，PC12细胞LC3自噬荧光强度最高；透射电镜也观察到酒精作用的PC12细胞质中自噬体和自噬溶酶体。Western blotting结果显示，不同浓度酒精处理PC12细胞2h，P62蛋白表达量显著增加 (P<0.01)；用200mmol/L浓度酒精处理PC12细胞，P62蛋白表达在2h达到最高值。 结论 酒精诱导PC12细胞的自噬作用，P62蛋白参与自噬调控过程。
Objective To utilize pheochromocytoma (PC12) cells to examine the effect of ethanol on autophagy and the role of P62 in ethanol-induced autophagy. Methods The inhibition effect of ethanol on proliferation of PC12 cells was examined by MTT assay. Indirect immunofluorescence was used to detect location of LC3, a biomarker of autophagy, and P62. The LC3 fluorescence intensity in cellular cytosol was measured using a high content screening imaging system. The ultrastructural morphology of autophagic vacuoles and autophagy-lysosome was observed under the transmission electron microscope. Protein expression of P62 was detected with Western blotting analysis. Results Compared to the control in serumfree medium after 24hours, ethanol at the concentrations of 100mmol/L, 200mmol/L, 400mmol/L and 800mmol/L decreased cell proliferation of PC12 cells to 91.97% (P<0.05)，72.63%(P<001)，58.23%(P<0.01) and 50.82%(P<0.01) respectively, indicating a dose-dependent inhibitory effect of ethanol on cell proliferation.Indirect immunofluorescence staining and the high content screening imaging system showed that ethanol increased LC3 fluorescence intensity and the co-localization of LC3 with P62 in PC12 cellular cytosol. The transmission electron microscope showed the ultrastructural morphology of autophagic vacuoles and autophagy-lysosome in PC12 cells incubated for 2hours in the presence of 200mmol/L ethanol. Compared with the control in serum-free medium, the protein expression of P62 increased significantly in different ethanol concentration treatment groups (P<0.01). The crest-time appeared at 2hours after the PC12 cells were treated with 200mmol/L ethanol. Conclusion Ethanol may induce autophagy of PC12 cells. P62 may be involved in the autophagy against ethanol-induced cytotoxicity.
酒精 / 自噬 / LC3 / P62 / PC12细胞 / 间接免疫荧光法 / 透射电镜
Ethanol / Autophagy / LC3 / P62 / PC12 cell / Indirect immunofluorescence / Transmission electron microscopy
［1］ Higgins GC，Devenish RJ，Beart PM，et al. Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death ［J］. Cell Mol Life Sci，2011，68(22)：3725-3740.
［2］Wang HJ，Tan YZh. Morphological features andfunctional implications of autophagy ［J］. Acta Anatomica Sinica，2009，40(5)：844-849.（in Chinese）
王海杰, 谭玉珍. 细胞自噬的形态学特征和功能意义［J］. 解剖学报, 2009, 40(5):844-849.
［3］Tizon B，Sahoo S，Yu H，et al. Induction of autophagy by cystatin C: a mechanism that protects murine primary cortical neurons and neuronal cell lines ［J］. PLoS One，2010，5(3)：e9819.
［4］Harper JW，Schulman BA. Structural complexity in ubiquitin recognition ［J］. Cell，2006，124(6)：1133-1136.
［5］Johansen T，Lamark T. Selective autophagy mediated by autophagic adapter proteins［J］. Autophagy，2011，7(3)：279-296.
［6］Pankiv S，Clausen TH，Lamark T，et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy ［J］. J Biol Chem，2007，282(33)：24131-24145.
［7］Feng AQ，Li YQ，Wang LZh, et al. Pro-apoptotic effect of ethanol on the expression and activity of N-SMase in PC12 cells ［J］. Journal of Medical Molecular Biology，2010，7(6)：476-482.（in Chinese）
冯爱芹，厉永强，王利枝，等. 酒精促PCI2细胞凋亡及对神经鞘磷脂合酶表达和活性的影响 ［J］. 医学分子生物学杂志，2010，7(6)：476-482.
［8］Sari Y，Weedman JM，Ge S. Activity-dependent neurotrophic factor-derived peptide prevents alcohol-induced apoptosis, in part, through Bcl2 and c-Jun N-terminal kinase signaling pathways in fetal brain of C57BL/6 mouse ［J］. Neuroscience，2012，202：465-473.
［9］Liu L，Cao JX，Sun B，et al. Mesenchymal stem cells inhibition of chronic ethanol-induced oxidative damage via upregulation of phosphatidylinositol-3 -kinase/Akt and modulation of extracellular signal-regulated kinase 1/2 activation in PC12 cells and neurons ［J］. Neuroscience，2010，167(4)：1115-1124.
［10］Sheth DS，Tajuddin NF，Druse MJ. Antioxidant neuroprotection against ethanol-induced apoptosis in HN2-5 cells［J］. Brain Res，2009，1285：14-21.
［11］Wu D，Wang X，Zhou R，et al. Alcohol steatosis and cytotoxicity: the role of cytochrome P4502E1 and autophagy［J］. Free Radic Biol Med，2012，53(6)：1346-1357.
［12］Ding WX，Manley S，Ni HM. The emerging role of autophagy in alcoholic liver disease ［J］. Exp Biol Med (Maywood)，2011，236(5)：546-556.
［13］Luo S，Chen Q，Cebollero E，et al. Mitochondria：one of the origins for autophagosomal membranes［J］? Mitochondrion，2009，9(4)：227-231.
［14］Lee HM，Shin DM，Yuk JM，et al. Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1［J］. J Immunol，2011, 186(2)：1248-1258.
［15］Harper JW，Schulman BA. Structural complexity in ubiquitin recognition ［J］.Cell，2006，124(6)：1133-1136.
［16］Eskelinen EL，Reggiori F，Baba M，et al. Seeing is believing:the impact of electron microscopy on autophagy research［J］. Autophagy，2011，7(9)：935-936.
［17］Pankiv S，Lamark T，Bruun JA，et al. Nucleocytoplasmic shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to promyelocytic leukemia bodies［J］. J Biol Chem，2010，285(8)：5941-5953.
［18］Komatsu M，Ichimura Y. Physiological significance of selective degradation of p62 by autophagy［J］. FEBS Lett，2010，584(7)：1374-1378.
省自然科学基金资助项目;国家自然科学基金资助项目
/
| 〈 |
|
〉 |
