Oct4基因转染对人牙髓细胞增殖和多向分化能力的影响  

Transient transfection of transcription factor Oct4 on the proliferation and multilineage differentiation of human dental pulp cells

作　　者：张芳[1] 刘路[1] 韦曦[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2013年第3期4-8,共5页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(81070830);广东省科技计划(2010B031600063);广东省科技计划国际合作项目(00272400127680054);广东省中医药局科研课题(20121147);中央高校基本科研业务费专项基金(11ykpy51)

摘　　要：目的研究腺病毒介导的转录因子Oct4转染人牙髓细胞(DPCs)对其增殖和多向分化能力的影响。方法以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)的腺病毒载体Ad5-Oct4-EGFP,筛选最佳感染复数(MOI)转染DPCs,检测其增殖及成牙本质、成脂分化能力的改变。结果采用Admax包装系统成功构建并获得高滴度的腺病毒载体,筛选最适MOI=200,CCK-8检测结果显示,Ad5-Oct4-EGFP组2d起吸光度(A)值高于对照组,7、14d与对照组相比差异有统计学意义(P<0.05),矿化诱导21d后,茜素红染色显示Ad5-Oct4-EGFP组与对照组相比,矿化结节数量多且体积最大,实时荧光定量反转录聚合酶链反应(RT-PCR)结果显示,DSPP、BSP和CollagenⅠmRNA的表达量高于对照组(P<0.05);成脂诱导后细胞胞体呈立方状,油红O染色显示Ad5-Oct4-EGFP组细胞浆内出现较多脂滴,脂肪特异性标志LPL和PPARγ2mRNA水平较对照组显著上调(P<0.05),Ad5-EGFP和空白对照组间差异无统计学意义(P>0.05)。结论转录因子Oct4可促进DPCs增殖和多向分化能力,改善DPCs的干性。Objective To investigate the effect of Ad5-Oct4-EGFP transfection on the proliferation and multilineage differentiation capabilities of human dental pulp cells（DPCs）. Methods The recombinant adenovirus was constructed by Admax system which carried transcription factor and enhanced green fluorescence protein enhanced green fluorescent protein （EGFP）, and the optimal multiplicity of infection （MOI） was selected. After transfection, CCK-8 was used to examine the proliferation of transfected cells at days 1, 2, 3, 5, 7 and 14. Transfected DPCs were cultured in the odontogenic and adipogenic induction media for 21 days respectively. Odontogenic and adipogenic differentiation was investigated by Alizarin Red staining and Oil Red O staining, and the odontogenic and adipogenic differentiation markers were evaluated by RT-PCR. Results The recombinant adenoviral vector of Oct4 gene was successfully constructed and the MOI value of 200 was selected as the optimal MOI for the further study. The CCK-8 test revealed that the OD value in Ad5-Oct4-EGFP transfected ceils was higher than that in the control groups at day 2, with significant increase at day 7 and day 14 （P 〈 0.05）. The results of Alizarin Red staining indicated that Oct4 gene transfection promoted more calcified nodules formation in Ad5-Oet4-EGFP group. DSPP, BSP and collagen I mRNA expression levels were significantly greater in Ad5-Oct4-EGFP transinfected cells than those of the control groups at days 21 （P〈 0.05）. Adipogenic differentiation was demonstrated by the large accumulation of neutral lipid vacuoles indicated by the Oil Red 0 stain in Ad5-Oct4-EGFP group, and further confirmed by the significant up-regulation of LPL and PPARγ2 mRNAs compared with the control groups （P 〈 0.05）. Conclusion Our study has demonstrated Oct4 can improve the proliferation and muhilineage differentiation capablity of cultured DPCs.

关 键 词：牙髓细胞 转录因子Oct4 增殖 多向分化 

分 类 号：R780.2[医药卫生—口腔医学]