摘要
目的确定低剂量力达霉素可诱导小鼠胚胎癌P19细胞分化,并探讨其可能的作用机制。方法体外培养P19细胞,分别加入力达霉素和维甲酸(retinoiacid,RA),观察细胞形态,并分别行PCR和免疫荧光染色检测巢蛋白(Nestin)和微管相关蛋白2(MAP2)的表达。Western blot检测低剂量力达霉素和维甲酸处理该细胞后Oct4的表达。分别用慢病毒Oct4和si-Oct4感染P19细胞,使其过表达和下调Oct4,观察细胞形态,并行PCR和免疫荧光染色观察Nestin和MAP2的变化。结果 P19细胞经低剂量力达霉素和维甲酸处理后,形态上出现神经细胞样突起,且PCR和免疫荧光染色均示Nestin和MAP2的表达增强。应用低剂量力达霉素和维甲酸处理P19细胞后,Oct4的表达下降。过表达Oct4的P19细胞经低剂量力达霉素处理后未出现神经细胞样突起,且Nestin和MAP2的表达未增强,下调Oct4的P19细胞未经低剂量力达霉素处理,出现与其相反的结果。结论低剂量力达霉素可以诱导P19细胞分化,并且该分化可能与转录因子Oct4下调相关。
Objective To determine the low dose lidamycin induced mouse embryonal carcinoma P19 cells differenti- ated into neuron like cells,and to explore its possible mechanism of action. Methods Cultured mouse embryonal carci- noma P19 cells were added 0.01nM Lidamycin and 0.5μM retinoic acid （retinoiacid, RA） treatment, respectively, ob- served at different time points （0d,2d,4d,6d,8d and 10d）two sets of changes in cell morphology. And two groups of cells were performed RT PCR to detect its nerve cell-specific markers Nestin and MAP2 mRNA levels,and immunoflu- orescence staining Nestin and MAP2 protein expression. Two groups of cells Oct4 protein expression were detected by Western blot. Used to express Oct4 infected with lentivirus P19 cells by lidamycin same inducing conditions were ob served at different time points changes in cell morphology,and were detected on the mRNA and protein levels by RT- PCR and immunofluorescence staining Nestin and the change of the MAP2. In contrast,the siRNA-Oct4 transfection to p19 cells following tune Oct4,given the same induction conditions to observe the changes in the different time points cell morphology of,and RT PCR and immunefluorescence staining, respectively,observed Nestin and MAP2 of mRNA and protein of changes. Results P19 cells were treated with low dose lidamycin gradually differentiate into neuron-like cells, the performance of the level of mRNA expression of Nestin of MAP2 expression is significantly upregulated im- munofluorescence detection obvious increase in the expression of these two proteins,the results with RT-PCR the re- sults are the same. P19 cells to do the same treatment with retinoic acid,a similar result. Oct4 expression was signifi- cantly decreased after the application of low dose lidamycin and retinoic acid treatment of P19 cells. P19 cells over-ex pression of Oct4 after low dose lidamycin processing phenomenon does not appear to differentiate into neuron-like ceils. Reduced the Oct4 gene in P19 cells after induction of differentiation cultu
出处
《中国实验诊断学》
2013年第2期222-225,F0003,共5页
Chinese Journal of Laboratory Diagnosis
关键词
低剂量力达霉素
分化
P19
OCT4
Low dose Lidamycin
differentiation
P19 cells
transcription factor Oct4
