构建基于Keima蛋白的细胞自噬评价体系
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摘要
目的:构建表达Cyto-Keima蛋白的宫颈癌细胞系HeLa及小鼠原代神经元,作为新的细胞自噬评价体系。方法:包装pCDH-Cyto-Keima慢病毒,感染HeLa细胞和小鼠原代神经元,采用激光共聚焦荧光显微镜观察Cyto-Kei?ma蛋白在HeLa细胞和小鼠原代神经元中的表达情况,用已知自噬诱导剂和抑制剂处理细胞,检测该体系对自噬的响应。结果:感染Cyto-Keima慢病毒48 h后,通过激光共聚焦荧光显微镜观察,细胞表达Cyto-Keima蛋白;在Earle平衡盐溶液或自噬诱导剂雷帕霉素处理下,HeLa-Cyto-Keima细胞和Cyto-Keima原代神经元自噬水平明显增强;在自噬抑制剂巴弗洛霉素A1和氯喹处理下,细胞自噬水平明显降低。结论:构建了基于Cyto-Keima蛋白的细胞自噬评价体系。
Objective:To establish an evaluation system for autophagy in cervical cancer HeLa cell line and mouse primary neurons based on Cyto-Keima protein.Methods:HeLa cells and mouse primary neurons were infected with Cyto-Keima lentivirus. Then the fluorescence was detected by confocal microscopy. We treated cells with autophagy inducers and inhibitors to estimate the change of Cyto-Keima fluorescence.Results:Cyto-Keima was expressed in HeLa cells and mouse primary neurons after lentivirus infection. Earle′s balanced salts and rapamycin, the well-known autophagy inducers, actived the autophagy activities in HeLa-Cyto-Keima cells and CytoKeima primary neurons. Autophagy inhibitors such as bafilomycin A1 and chloroquine inhibited autophagy of HeLaCyto-Keima cells and Cyto-Keima primary neurons.Conclusion:We successfully established an evaluation system for autophagy based on Cyto-Keima protein in HeLa cells and primary neurons.
Objective:To establish an evaluation system for autophagy in cervical cancer HeLa cell line and mouse primary neurons based on Cyto-Keima protein.Methods:HeLa cells and mouse primary neurons were infected with Cyto-Keima lentivirus. Then the fluorescence was detected by confocal microscopy. We treated cells with autophagy inducers and inhibitors to estimate the change of Cyto-Keima fluorescence.Results:Cyto-Keima was expressed in HeLa cells and mouse primary neurons after lentivirus infection. Earle′s balanced salts and rapamycin, the well-known autophagy inducers, actived the autophagy activities in HeLa-Cyto-Keima cells and CytoKeima primary neurons. Autophagy inhibitors such as bafilomycin A1 and chloroquine inhibited autophagy of HeLaCyto-Keima cells and Cyto-Keima primary neurons.Conclusion:We successfully established an evaluation system for autophagy based on Cyto-Keima protein in HeLa cells and primary neurons.
引文
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[2]Mizushima N.A brief history of autophagy from cell biology to physiology and disease[J].Nat Cell Biol,2018,20(5):521-527.
[3]Gatica D,Lahiri V,Klionsky D J.Cargo recognition and degradation by selective autophagy[J].Nat Cell Biol,2018,20(3):233-242.
[4]Mowers E E,Sharifi M N,Macleod K F.Autophagy in cancer metastasis[J].Oncogene,2017,36(12):1619-1630.
[5]Kiriyama Y,Nochi H.The function of autophagy in neurodegenerative diseases[J].Int J Mol Sci,2015,16(11):26797-26812.
[6]Fougeray S,Pallet N.Mechanisms and biological functions of autophagy in diseased and ageing kidneys[J].Nat Rev Nephrol,2015,11(1):34-45.
[7]苑杰,江沛,吕佩源,等.细胞自噬与疾病发生发展的关系及作为治疗靶点的研究进展[J].医学研究生学报,2018,(1):88-91.200
[8]Katayama H,Kogure T,Mizushima N,et al.A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery[J].Chem Biol,2011,18(8):1042-1052.
[9]Campos T,Ziehe J,Palma M,et al.Rheb promotes cancer cell survival through p27Kip1-dependent activation of autophagy[J].Mol Carcinog,2016,55(2):220-229.
[10]Takeda A,Tamano H.Regulation of extracellular Zn(2+)homeostasis in the hippocampus as a therapeutic target for Alzheimer′s disease[J].Expert Opin Ther Targets,2015,19(8):1051-1058.