Gene expression profiles of DFCs and SHED 48 hours after in vitro transfection with a TP53 plasmid, a SP1 plasmid, or an empty vector.. Homo sapiens

Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35 % of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs. Overall design: Total RNAs were isolated from dental follicle cells after 48 hours of transfection with a TP53 expressions plasmid, a SP1 expressions plasmid and for control with an empty vector.

牙囊（Dental follicle）是一类包绕发育中牙齿的疏松结缔组织。牙囊细胞（Dental follicle cells, DFCs）在牙周与骨再生等组织工程应用中具备良好的应用潜力。然而，目前学界对其成骨分化背后的分子机制仍知之甚少。在前期研究中，我们发现牙囊细胞成骨分化过程中受调控的基因中，有超过35%的基因启动子区域存在转录因子TP53与SP1的结合位点。但目前这类转录因子在牙源性干细胞中的作用仍未明确。我们提出假说：这两种转录因子均可调控牙源性干细胞的细胞增殖与分化过程。因此，我们将针对这两种转录因子的过表达载体，瞬时转染至牙囊细胞与人脱落乳牙干细胞（Stem cells from human exfoliated deciduous teeth, SHED）中。在过表达SP1与TP53后，两种牙源性细胞中SP1均可调控细胞增殖，TP53则可调控成骨分化。采用小干扰RNA（small interfering RNA, siRNA）介导沉默TP53与SP1后，上述对细胞增殖与分化的调控效应则相对减弱。这表明我们在TP53与SP1过表达后观测到的调控效应属于间接作用，且受到复杂的调控网络调控。有趣的是，TP53过表达后牙囊细胞中上调的生物学过程，与SP1过表达后脱落乳牙干细胞中下调的生物学过程高度相似。此类受调控的生物学过程涉及细胞运动、创伤愈合与程序性细胞死亡。综上，本研究证实SP1与TP53均可调控脱落乳牙干细胞与牙囊细胞的细胞增殖、分化及相似的生物学过程。实验设计：将牙囊细胞分别转染TP53过表达质粒、SP1过表达质粒，并以空质粒作为对照，转染48小时后提取总RNA。