Transient transfection of transcription factor Oct4 on the expressions of pluripotencyassociated genes in human dental pulp cells

doi:10.3877/cma.j.issn.1674-1366.2011.03.003

Abstract

Abstract:

Objective

To investigate the effect of Ad5-Oct4-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-Oct4-EGFP transfer. The mRNA expressions of Oct4 AP Nanog Utf1 Sox2 were detected in the transfected 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 Utf1 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 Utf1 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 pluripotency-associated genes in DPCs and may influence their developmental potency.

Key words: Dental pulp cells, Transcription factor Oct4, Pluripotency-associated genes

Xi WEI, Fang ZHANG, Lu LIU, Xiaoyan ZHOU, Qian-ying KONG. Transient transfection of transcription factor Oct4 on the expressions of pluripotencyassociated genes in human dental pulp cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2011, 5(04): 347-355.

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URL: https://zhkqyxyjzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1366.2011.03.003

https://zhkqyxyjzz.cma-cmc.com.cn/EN/Y2011/V5/I04/347

Figures/Tables 9

References 23

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基因	引物序列
Oct4	Forward Primer：5'-GCAACCTGGAGAATTTGTTCCT-3'
	Reverse Primer：5'-CCACACTCGGACCACATCCT-3'
AP	Forward Primer：5'-GCAACTTCCAGACCATTGGC-3'
	Reverse Primer：5'-TCCCACTGACTTCCCTGCTT-3'
Nanog	Forward Primer：5'-GCCTCACACGGAGACTGTCTC-3'
	Reverse Primer：5'-AGTGGGTTGTTTGCCTTTGG-3'
Utf1	Forward Primer：5'-ACAAGTTCCTTAAAGACAAGTTTCGC-3'
	Reverse Primer：5'-GATCTGCTCGTCGAAGGGC-3'
Sox2	Forward Primer：5'-GAGAACCCCAAGATGCACAAC-3'
	Reverse Primer：5'-CGCTTAGCCTCGTCGATGA-3'
β-actin	Forward Primer：5'-GCGCGGCTACAGCTTCA-3'
	Reverse Primer：5'-TCTCCTTAATGTCACGCACGAT-3'

基因	引物序列
Oct4	Forward Primer：5'-GCAACCTGGAGAATTTGTTCCT-3'
	Reverse Primer：5'-CCACACTCGGACCACATCCT-3'
AP	Forward Primer：5'-GCAACTTCCAGACCATTGGC-3'
	Reverse Primer：5'-TCCCACTGACTTCCCTGCTT-3'
Nanog	Forward Primer：5'-GCCTCACACGGAGACTGTCTC-3'
	Reverse Primer：5'-AGTGGGTTGTTTGCCTTTGG-3'
Utf1	Forward Primer：5'-ACAAGTTCCTTAAAGACAAGTTTCGC-3'
	Reverse Primer：5'-GATCTGCTCGTCGAAGGGC-3'
Sox2	Forward Primer：5'-GAGAACCCCAAGATGCACAAC-3'
	Reverse Primer：5'-CGCTTAGCCTCGTCGATGA-3'
β-actin	Forward Primer：5'-GCGCGGCTACAGCTTCA-3'
	Reverse Primer：5'-TCTCCTTAATGTCACGCACGAT-3'

MOI	Ad5-Oct4-EGFP
1	1.240 ± 2.333
10	15.730 ± 1.358
100	40.300 ± 3.588
200	86.100 ± 1.633
500	91.100 ± 1.245

MOI	Ad5-Oct4-EGFP
1	1.240 ± 2.333
10	15.730 ± 1.358
100	40.300 ± 3.588
200	86.100 ± 1.633
500	91.100 ± 1.245

转染时间（d）	Oct4	AP	Nanog	Utf1
对照组	0.0471 ± 0.0012	0.0346 ± 0.0015	0.0021 ± 0.0009	0.0009 ± 0.0004
1	0.0675 ± 0.0173	0.0552 ± 0.0033	0.0257 ± 0.0045	0.0168 ± 0.0004
2	0.1035 ± 0.0302	0.0083 ± 0.0011	0.0104 ± 0.0003	0.0004 ± 0.0002
3	0.1238 ± 0.0023	0.0341 ± 0.0032	0.0891 ± 0.0102	0.0050 ± 0.0004
5	0.2403 ± 0.0072	0.0207 ± 0.0023	0.0585 ± 0.0137	0.0018 ± 0.0003
7	0.2487 ± 0.0469	2.4333 ± 0.3061	1.0531 ± 0.1055	0.6753 ± 0.0681
14	0.1917 ± 0.0231	0.0251 ± 0.0017	0.0886 ± 0.0093	0.0013 ± 0.0001

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