Oct4基因转染对人牙髓细胞多能相关转录因子表达的影响

Transient transfection of transcription factor Oct4 on the expressions of pluripotency-associated genes in human dental pulp cells

目的研究腺病毒介导的转录因子Oct4转染人牙髓细胞(DPCs)对其多能相关转录因子表达的影响。方法采用Admax包装系统构建携带目的基因Oct4和增强型绿色荧光蛋白(EGFP)报告基因的腺病毒载体,以不同感染复数(MOI)转染DPCs,荧光倒置显微镜检测转染率以最适MOI转染DPCs,实时荧光定量RT-PCR检测转染后1、2、3、5、7和14d,Oct4、AP、Nanog、Utf1和Sox2的表达变化。结果利用Admax包装系统成功构建并获得高滴度重组腺病毒载体,最适MOI=200,实时荧光定量RT-PCR检测结果发现,Oct4、AP、Nanog和Utf1 mRNA在转染后1d开始表达,7d达到最大值,与对照组相比有统计学意义(P<0.05),随后表达下调,且7d时AP mRNA的表达量显著高于Oct4、Utf1和Nanog(P<0.05),Sox2在转染后各时间点均不表达。结论转录因子Oct4瞬时转染可激活多能相关转录因子AP、Nanog和Utf1的表达,从而可能影响DPCs的未分化性能。

Objective To investigate the effect of Ad5-Oet4-EGFP transfection on the pluripotency- associated genes expressions of human dental pulp cells （DPCs）. Methods The recombinant adenovirus was constructed by Admax system, which carried transcription factor Oct4 and enhanced green fluorescence protein （EGFP）. After transfected with different multiplicities of infection （MOI） for 48 hours, the transfection efficiency was calculated under fluorescence microscope and optimal MOI was selected for this Ad5-Oet4-EGFP transfer. The mRNA expressions of Oct4 AP Nanog Utfl Sox2 were detected in the transfeeted cells at days 1, 2, 3, 5, 7 and 14 by RT-PCR. Results The recombinant adenoviral vector of Oct4 gene was successfully constructed and high titer of the recombinant adenovirus was obtained. The MOI value of 200 was selected as the optimal MOI for the further study. The mRNA levels of Oct4 Utfl Nanog AP were expressed at day 1 in Ad5-Oct4-EGFP transfected group, reaching the peak value at day 7 （P 〈 0.05） and then decreased along time. AP mRNA level was significantly greater than Oct4 Utfl and Nanog at day 7 （P〈 0.05）. Expression of Sox2 mRNA were not detected. Conclusions Our study has demonstrated that Oct4 can activate the key pluripoteney-associated genes in DPCs and may influence their developmental potency.