转录因子Nanog过表达对人牙髓细胞增殖及多向分化能力的影响

doi:10.3877/cma.j.issn.1674-1366.2015.05.002

目的

探讨过表达多能相关转录因子Nanog对人牙髓细胞增殖及多向分化能力的影响。

方法

构建过表达Nanog的慢病毒载体质粒pSIN-EF2-Nanog-IRES-GFP-puro，采用psPAX和pMD2.G慢病毒包装系统转入293T细胞进行病毒液的生产和收取，转染生长良好的第3代人牙髓细胞，构建稳定过表达Nanog的人牙髓细胞株，以空载体转染的细胞为对照组，对细胞进行成脂及成牙本质诱导。采用BrdU法检测细胞的增殖情况，检测多能性转录因子Oct4和Sox2的表达，以及细胞成脂、成牙本质分化能力。采用单因素方差分析数据。

结果

成功构建Nanog过表达的人牙髓细胞株；BrdU法检测结果显示，Nanog过表达组比对照组细胞增殖能力强（F = 90.421，P = 0.000）。过表达组Oct4、Sox2 mRNA和蛋白表达水平明显高于对照组（F_{Oct4 mRNA} = 71.649，P_{Oct4 mRNA}=0.000；F_{Sox2 mRNA} = 106.278，P_{Sox2 mRNA} = 0.000；F_Oct4蛋白 = 41.632，P_Oct4蛋白 = 0.002；F_Sox2蛋白 = 38.962，P_Sox2蛋白 = 0.002）。在矿化诱导条件下，Nanog过表达组DSPP、DMP1和OCN表达量、矿化结节产生量高于对照组（F_DSPP = 15.261，P_DSPP<0.05；F_DMP1 = 16.235，P_DMP1<0.05；F_OCN = 17.019，P_OCN<0.05）；成脂诱导21 d后，Nanog过表达组LPL和PPARγ2表达量及脂质产生高于对照组（F_LPL = 15.542，P_LPL<0.05；F_PPARγ2 = 10.437，P_PPARγ2<0.05）。

结论

过表达Nanog能提高人牙髓细胞增殖能力，促进多能性转录因子Oct4、Sox2的表达，增强细胞多向分化能力。

关键词: 牙髓细胞, Nanog, 细胞增殖, 多向分化能力, 多能相关转录因子

Objective

To investigate the effect of overexpression Nanog on the cell proliferationand multipotent differentiation potentiality of human dental pulp cells (hDPCs) .

Methods

Primary human pulp cells were transfected with plasmid pSIN-EF2-Nanog-IRES-GFP-puro by lentiviral transfection and stable nanog-expressing cell line was screened under fluorescence microscope. The effect of Nanog on the proliferation of hDPCs was examined with BrdU assay. The expressions levels of pluripotency factor Oct4 and Sox2 were detected using real-time PCR and western blotting. Nanog-hDPCs were cultured in odontogenic and adipogenic induction media respectively. The odontogenic and adipogenic differentiation potentiality were evaluated by Alizarin red S staining and Oil red O staining, and the odontogenic and adipogenic markers were detected using real-time PCR.

Results

The recombinant lentiviral vector containing Nanog was successfully constructed and showed high efficiency during infection in hDPCs. The BrdU assay showed that the growth rate of Nanog-hDPCs was higher compared with the controls (F = 90.421, P = 0.000) . The expressions of Oct4 and Sox2 were increased in Nanog-hDPCs in comparison with the controls (F_{Oct4 mRNA}= 71.649, P_{Oct4 mRNA} = 0.000; F_{Sox2 mRNA}= 106.278, P_{Sox2 mRNA} = 0.000; F_Oct4 = 41.632, P_Oct4 = 0.002; F_Sox2 = 38.962, P_Sox2 = 0.002) . Furthermore, the mRNA levels of DSPP, DMP1, and OCN were increased, and the number of mineralized nodules was significantly higher in Nanog-hDPCs (F_DSPP = 15.261, P_DSPP < 0.05; F_DMP1 = 16.235, P_DMP1 < 0.05; F_OCN = 17.019, P_OCN < 0.05) . Meanwhile, significantly higher mRNA expression levels of LPL and PPARγ2 and more lipid droplets were observed in Nanog-hDPCs compared to the controls (F_LPL = 15.542, P_LPL < 0.05; F_PPARγ2 = 10.437, P_PPARγ2 < 0.05) .

Conclusion

Nanog overexpression enhanced cell proliferation, upregulated the expression of pluripotent markers, and promoted the multipotent differentiation potential (odontogenic and adipogenic differentiation) of hDPCs.

Key words: Dental pulp cells, Nanog, Cell proliferation, Multipotent differentiation, Pluripotency-associated transcription factors

表1 实时荧光定量PCR引物序列

基因	引物序列
Nanog	F:5′-GATTTGTGGGCCTGAAGAAA-3′
?	R:5′-ATGGAGGAGGGAAGAGGAGA-3′
Oct4	F:5′-GTGGAGGAAGCTGACAACAA-3′
?	R:5′-GGTTCTCGATACTGGTTCGC-3′
Sox2	F:5′-AACCCCAAGATGCACAACTC-3′
?	R:5′-GCTTAGCCTCGTCGATGAAC-3′
DSPP	F:5′-GCCACTTTCAGTCTTCAA AGAGA-3′
?	R:5′-GCCCAAATGCAAAAATATGTA-3′
DMP1	F:5′-TGGGCATAGATTTCCTCTTTG-3′
?	R:5′-TGAGCAGGATGCTGATCTTC-3′
OCN	F:5′-CTCACACTCCTCGCCCTATT-3′
?	R:5′-TTGGACACAAAGGCTGCAC-3′
LPL	F:5′-CCCCGAGATGGAGAGCAAAG-3′
?	R:5′-ATCTCTTCTTTGGTCGGCGG-3′
PPARγ2	F:5′-GCAAACCCCTATTCCATGCTG-3′
?	R:5′-CACGGAGCTGATCCCAAAGT-3′
GAPDH	F:5′-AAGGTGAAGGTCGGAGTCAA-3′
?	R:5′-AATGAAGGGGTCATTGATGG-3′

表1 实时荧光定量PCR引物序列

基因	引物序列
Nanog	F:5′-GATTTGTGGGCCTGAAGAAA-3′
?	R:5′-ATGGAGGAGGGAAGAGGAGA-3′
Oct4	F:5′-GTGGAGGAAGCTGACAACAA-3′
?	R:5′-GGTTCTCGATACTGGTTCGC-3′
Sox2	F:5′-AACCCCAAGATGCACAACTC-3′
?	R:5′-GCTTAGCCTCGTCGATGAAC-3′
DSPP	F:5′-GCCACTTTCAGTCTTCAA AGAGA-3′
?	R:5′-GCCCAAATGCAAAAATATGTA-3′
DMP1	F:5′-TGGGCATAGATTTCCTCTTTG-3′
?	R:5′-TGAGCAGGATGCTGATCTTC-3′
OCN	F:5′-CTCACACTCCTCGCCCTATT-3′
?	R:5′-TTGGACACAAAGGCTGCAC-3′
LPL	F:5′-CCCCGAGATGGAGAGCAAAG-3′
?	R:5′-ATCTCTTCTTTGGTCGGCGG-3′
PPARγ2	F:5′-GCAAACCCCTATTCCATGCTG-3′
?	R:5′-CACGGAGCTGATCCCAAAGT-3′
GAPDH	F:5′-AAGGTGAAGGTCGGAGTCAA-3′
?	R:5′-AATGAAGGGGTCATTGATGG-3′

图1 Nanog过表达人牙髓细胞株的构建

图2 过表达Nanog人牙髓细胞的增殖能力

图3 过表达Nanog对人牙髓细胞表达Oct4、Sox2的影响

图4 过表达Nanog对人牙髓细胞成脂及成牙本质能力的影响

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Effects of Nanog overexpression on the proliferation and multipotent differentiation potentiality of human dental pulp cells

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Effects of Nanog overexpression on the proliferation and multipotent differentiation potentiality of human dental pulp cells