TrAmplification of Human Dental Follicle Cells by piggyBac Transposon - Mediated Reversible Immortalization System.
TrAmplification of Human Dental Follicle Cells by piggyBac Transposon - Mediated Reversible Immortalization System.
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通过piggyBac转座子介导的可逆永生化系统对人牙囊细胞进行Tr扩增
DOI：
10.1371/journal.pone.0130937
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Deng F
中科院分区：
文献类型：
--
作者：
Wu Y;Feng G;Song J;Zhang Y;Yu Y;Huang L;Zheng L;Deng F
牙囊细胞是牙周组织的前体细胞。在一定的分化条件下，DFCs可以被诱导分化为软骨细胞、成骨细胞和成脂细胞。然而，DFC在体外的寿命有限，因此很难获得足够的细胞用于基础研究和翻译应用。PMPH86是一种含SV40T-Ag盒的转座子载体，可被FRT重组酶去除。在这里，我们证明了pMPH86可以通过可逆永生化有效地扩增人DFCs。永生化的DFC(IDFC)具有较高的增殖活性，经FLP重组酶去永生化后，其增殖活性可逆转至永生化前的水平。成骨细胞和去永生化的成骨细胞(DDFC)在体外表达大多数DFC标志物，在BMP9的诱导下，经基因表达和蛋白标记证实，它们可以分化为软骨、成骨和成脂细胞，并保持多向分化潜能。我们还证明了iDFCs的端粒酶活性显著升高并维持在较高水平，而原代DFCs的端粒酶活性相对较低，并随着每一代的传代而下降。去除SV40T-Ag使细胞永生化后，端粒酶活性降至永生化前的水平，随着传代次数的增加，端粒酶活性下降，与原代DFC相同。这些结果表明，猪胚胎永生化系统可能是一种潜在的原代细胞扩增策略，这对再生研究和进一步的临床应用是至关重要的。
Dental follicle cells (DFCs) are the precursor cells of periodontium. Under certain differentiation conditions, DFCs can be induced to differentiate into chondrogenic, osteogenic and adipogenic cells. However, DFCs has limited lifespan in vitro, so it’s difficult to harvest enough cells for basic research and translational application. pMPH86 is a piggyBac transposon-mediated vector which contains SV40 T-Ag cassette that can be removed by flippase recognition target (FRT) recombinase. Here we demonstrated the pMPH86 can effectively amplify human DFCs through reversible immortalization. The immortalized DFCs (iDFCs) exhibit higher proliferate activity, which can be reversed to its original level before immortalization when deimmortalized by FLP recombinase. The iDFCs and deimmortalized DFCs (dDFCs) express most DFC markers and maintain multiple differentiation potential in vitro as they can be induced by BMP9 to differentiate into chondrogenic, osteogenic and adipogenic cells evidenced by gene expression and protein marker. We also proved telomerase activity of iDFCs are significantly increased and maintained at a high level, while the telomerase activity of primary DFCs was relatively low and decreased with every passage. After SV40 T-Ag was removed to deimmortalize the cells, telomerase activity was reduced to its original level before immortalization and decreased with passages just the same as primary DFCs. These results suggest that piggyBac immortalization system could be a potential strategy to amplify primary cells, which is critical for regenerative research and further clinical application.