关键词: 白血病, 单核细胞, 急性, 细胞系, 肿瘤, 十四酰佛波乙酯, 自噬, 模型, 生物学

Abstract: Objective  To optimized the conditions of PMA to stimulate THP-1 cell differentiation in order to establish a cell model of autophagy, which provides a scientific basis for the researches related to cell autophagy.  Methods  THP-1 cells differentiated into macrophages induced by PMA.Fusion protein pcDNA3.1-YFP-LC3 plasmid was transferred into THP-1 cell, and YFP(Yellow fluorescent protein) can trace the formation of autophagosome. Morphological changes of differentiated cells were photographed by inverted microscope respectively at diffe-rent PMA concentrations（0，10，20，50，100，200 ng/ml） and different times（0，24，48，60 h）. LC3 protein was traced by fluorescence microscopy in different conditions. The mRNA expression levels of autophagy-related genes were detected by RT-qPCR（reverse transcription-quantitative polymerase chain reaction ）. SPSS 17.0 was used to do statistical analyze, the relative amount 2^-ΔΔCt of mRNA was showed as “x±s”, and the difference between gene expression was analyzed by Ct value. Paired t-test (P＜0.5) was used to analyse the expression of mRNA induced by EBSS(earle’s balanced salts solution). Results  When the PMA concentration was 100 ng/ml and the induction time was 24-48 h, the state of THP-1 derived cells reached best condition. After deal with EBSS medium, the formulation of YFP-LC3 autophagy in THP-1 derived cells was enhanced, and the expression levels of autophagy-rela-ted genes LC3、Atg5、Atg7、Beclin1 were increased at the same time, which indicated that autophagy cell model was successfully constructed (2^-ΔΔCt value were 1.35±0.16,1.18±0.39,1.44±0.12,1.08±0.09，while t value were 4.00,2.90,5.16,3.57，P＜0.05). Conclusion  The optimization culture conditions were 100 ng/ml PMA and 24-48 h induced time. Based on this condition, an ideal autophagy model was established, which providing a solid scientific foundation for autophagy-related research.

Key words: Leukemia, monocytic, acute, Cell line, tumor, Tetradecanoylphorbol acetate, Autophagy, Models, biological