ephrinB2基因转染脱落乳牙来源的牙髓干细胞提高其成骨/成牙本质分化的研究
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摘要
目的观察促红细胞生成素肝细胞激酶受体B4/促红细胞生成素肝细胞激酶受体配体B2 (EphB4/ephrinB2)信号通路在调控过表达ephrinB2的脱落乳牙来源牙髓干细胞(SHEDs)成骨/成牙本质分化中的作用。方法从滞留乳牙中分离出SHEDs,用hEfnB2-GFP-Bsd载体和空载体(GFP-Bsd)转染后进行成骨/成牙本质分化诱导。用反转录聚合酶链反应(RT-PCR)、碱性磷酸酶实验(ALP)实验、茜素红S染色实验来检测EfnB2-SHEDs组、空载体对照组(Vector-SHEDs)的成骨分化效果,并用Western blot、免疫沉淀和免疫共沉淀试验来检测ephrinB2和EphB4磷酸化蛋白表达水平。结果 ALP实验和茜素红S染色实验证实:EfnB2-SHEDs组比Vector-SHEDs组表现出更高的ALP活性和矿化能力。RT-PCR显示:EfnB2-SHEDs组成牙本质/成骨标志物的表达明显高于Vector-SHEDs组,在成骨分化中,EphB4受体通过磷酸化被活化。结论基因转染ephrinB2能够通过刺激ephrinB2和EphB4的磷酸化来提高SHEDs的成骨/成牙本质分化。
Objective To investigate the role of ephrinB2/EphB4 signaling in the osteogenic/odontogenic differentiation of SHEDs via over-expression ephrinB2 in SHEDs. Methods SHEDs were isolated from exfoliated deciduous teeth and transfected with hEfnB2-GFP-Bsd(GFP-Bsd as empty Vector). The transfected SHEDs were then induced into osteo-/odontoblasts under special differentiation medium. RT-PCR, Alizarin-red S staining, and ALP assay were used to analyze the effect of osteogenic/odontogenic differentiation of EfnB2-SHEDs and Vector-SHEDs. The phosphorylated protein expression level of ephrinB2 and EphB4 were invested via Western blot, immunoprecipitation assays and co-immunoprecipitation assays. Results Compared with Vector-SHEDs, EfnB2-SHEDs possessed higher ALP activity and mineralization capacity. The odonto-/osteogenic genes expression were significantly enhanced in EfnB2-SHEDs group, and the activity of EphB4 receptor was triggered through phosphorylation in odonto-/osteogenic differentiation. Conclusion Our study shows that the odonto-/osteogenic differentiation of SHEDs can be improved through stimulation of the phosphorylation of ephrinB2/Ephb4.
Objective To investigate the role of ephrinB2/EphB4 signaling in the osteogenic/odontogenic differentiation of SHEDs via over-expression ephrinB2 in SHEDs. Methods SHEDs were isolated from exfoliated deciduous teeth and transfected with hEfnB2-GFP-Bsd(GFP-Bsd as empty Vector). The transfected SHEDs were then induced into osteo-/odontoblasts under special differentiation medium. RT-PCR, Alizarin-red S staining, and ALP assay were used to analyze the effect of osteogenic/odontogenic differentiation of EfnB2-SHEDs and Vector-SHEDs. The phosphorylated protein expression level of ephrinB2 and EphB4 were invested via Western blot, immunoprecipitation assays and co-immunoprecipitation assays. Results Compared with Vector-SHEDs, EfnB2-SHEDs possessed higher ALP activity and mineralization capacity. The odonto-/osteogenic genes expression were significantly enhanced in EfnB2-SHEDs group, and the activity of EphB4 receptor was triggered through phosphorylation in odonto-/osteogenic differentiation. Conclusion Our study shows that the odonto-/osteogenic differentiation of SHEDs can be improved through stimulation of the phosphorylation of ephrinB2/Ephb4.
引文
[1] Liu J, Yu F, Sun Y, et al. Concise reviews: characteristics and potential applications of human dental tissue-derived mesenchymal stem cell[J]. Stem Cells,2015,33(3):627-38.
[2] Tierney E G, McSorley K, Hastings C L, et al. High levels of ephrinB2 overexpression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix[J]. J Control Release,2013,165:173-82.
[3] Lin Z, Rios H F, Cochran D L. Emerging regenerative approaches for periodontal reconstruction: a systematic review from the AAP regeneration workshop[J]. J Periodontol,2015,86 (2 Suppl):134–52.
[4] Heng B C, Wang S, Gong T, et al. EphrinB2 signaling enhances osteogenic/odontogenic differentiation of human dental pulp stem cells[J]. Arch Oral Biol,2018,87:62-71.
[5] Toda H, Yamamoto M, Uyama H, et al. Effect of hydrogel elasticity and ephrinB2-immobilized manner on Runx2 expression of human mesenchymal stem cells[J]. Acta Biomater,2017,58:312–22.
[6] Tonna S, Takyar F M, Vrahnas C, et al. EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis[J]. FASEB J, 2014,28(10):4482–96.
[7] Takyar F M, Tonna S, Ho P W, et al. EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone[J]. J Bone Miner Res,2013,28(4):912–25.
[8] Zhu S Y, Wang P L, Liao C S, et al. Transgenic expression of ephrinB2 in periodontal ligament stem cells (PDLSCs) modulates osteogenic differentiation via signaling crosstalk between ephrinB2 and EphB4 in PDLSCs and between PDLSCs and pre-osteoblasts within co-culture[J]. J Periodontal Res, 2017,52(3):562-73.
[2] Tierney E G, McSorley K, Hastings C L, et al. High levels of ephrinB2 overexpression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix[J]. J Control Release,2013,165:173-82.
[3] Lin Z, Rios H F, Cochran D L. Emerging regenerative approaches for periodontal reconstruction: a systematic review from the AAP regeneration workshop[J]. J Periodontol,2015,86 (2 Suppl):134–52.
[4] Heng B C, Wang S, Gong T, et al. EphrinB2 signaling enhances osteogenic/odontogenic differentiation of human dental pulp stem cells[J]. Arch Oral Biol,2018,87:62-71.
[5] Toda H, Yamamoto M, Uyama H, et al. Effect of hydrogel elasticity and ephrinB2-immobilized manner on Runx2 expression of human mesenchymal stem cells[J]. Acta Biomater,2017,58:312–22.
[6] Tonna S, Takyar F M, Vrahnas C, et al. EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis[J]. FASEB J, 2014,28(10):4482–96.
[7] Takyar F M, Tonna S, Ho P W, et al. EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone[J]. J Bone Miner Res,2013,28(4):912–25.
[8] Zhu S Y, Wang P L, Liao C S, et al. Transgenic expression of ephrinB2 in periodontal ligament stem cells (PDLSCs) modulates osteogenic differentiation via signaling crosstalk between ephrinB2 and EphB4 in PDLSCs and between PDLSCs and pre-osteoblasts within co-culture[J]. J Periodontal Res, 2017,52(3):562-73.