酒精诱导嗜铬细胞瘤细胞自噬及其与P62作用的关系

doi:10.3969/j.issn.0529-1356.2013.06.008

解剖学报 ›› 2013, Vol. 44 ›› Issue (6 ): 772-777.doi: 10.3969/j.issn.0529-1356.2013.06.008

酒精诱导嗜铬细胞瘤细胞自噬及其与P62作用的关系

董越娟¹ 石贞玉¹ 刘诗濛² 张维娟² 皇甫超申² 邓锦波¹刘彬^1*

1. 河南大学护理学院神经生物学研究所; 2. 河南大学医学院生物化学与分子生物学教研室，河南开封 475004

收稿日期:2013-02-26 修回日期:2013-07-09 出版日期:2013-12-06 发布日期:2013-12-06
通讯作者: 刘彬 E-mail:lbgood5912z@sina.com
基金资助:
省自然科学基金资助项目;国家自然科学基金资助项目

Effects of ethanol on autophagy and the role of P62 in ethanol-induced autophagy in pheochromocytoma cells

DONG Yue-juan¹ SHI Zhen-yu¹ LIU Shi-meng² ZHANG Wei-juan² HUANGFU Chao-shen² DENG Jin-bo¹ LIU Bin^1*

1. Institute of Neurobiology，College of Nursing of Henan University, He’nan Kaifeng 475004, China; 2. Department of Biochemistry and Molecule Biology, Medical College of Henan University, He’nan Kaifeng 475004, China

Received:2013-02-26 Revised:2013-07-09 Online:2013-12-06 Published:2013-12-06
Contact: LIU Bin E-mail:lbgood5912z@sina.com

摘要/Abstract

摘要：

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。

关键词: 酒精, 自噬, LC3, P62, PC12细胞, 间接免疫荧光法, 透射电镜

Abstract:

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 serumfree 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.

Key words: Ethanol, Autophagy, LC3, P62, PC12 cell, Indirect immunofluorescence, Transmission electron microscopy

中图分类号:

R737.1

董越娟石贞玉刘诗濛张维娟皇甫超申邓锦波刘彬* . 酒精诱导嗜铬细胞瘤细胞自噬及其与P62作用的关系[J]. 解剖学报, 2013, 44(6 ): 772-777.

DONG Yue-juan SHI Zhen-yu LIU Shi-meng ZHANG Wei-juan HUANGFU Chao-shen DENG Jin-bo LIU Bin*. Effects of ethanol on autophagy and the role of P62 in ethanol-induced autophagy in pheochromocytoma cells[J]. AAS, 2013, 44(6 ): 772-777.

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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

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