Mouse dental papilla cells(mDPCs)are cranial neural crest-derived dental mesenchymal cells that give rise to dentin-secreting odontoblasts after the bell stage during odontogenesis.The odontoblastic differentiation of...Mouse dental papilla cells(mDPCs)are cranial neural crest-derived dental mesenchymal cells that give rise to dentin-secreting odontoblasts after the bell stage during odontogenesis.The odontoblastic differentiation of mDPCs is spatiotemporally regulated by transcription factors(TFs).Our previous work reveals that chromatin accessibility was correlated with the occupation of the basic leucine zipper TF family during odontoblastic differentiation.However,the detailed mechanism by which TFs regulate the initiation of odontoblastic differentiation remains elusive.Here,we report that phosphorylation of ATF2(p-ATF2)is particularly increased during odontoblastic differentiation in vivo and in vitro.ATAC-seq and p-ATF2 CUT&Tag experiments further demonstrate a high correlation between p-ATF2 localization and increased chromatin accessibility of regions near mineralization-related genes.Knockdown of Atf2 inhibits the odontoblastic differentiation of mDPCs,while overexpression of p-ATF2 promotes odontoblastic differentiation.ATAC-seq after overexpression of p-ATF2 reveals that p-ATF2 increases the chromatin accessibility of regions adjacent to genes associated with matrix mineralization.Furthermore,we find that p-ATF2 physically interacts with and promotes H2BK12 acetylation.Taken together,our findings reveal a mechanism that p-ATF2 promotes odontoblastic differentiation at initiation via remodeling chromatin accessibility and emphasize the role of the phosphoswitch model of TFs in cell fate transitions.展开更多
目的观察矿物三氧化物凝聚体(Mineral Trioxide Aggregate,MTA)和三种改良盖髓剂(nRoot、Vitapex、iRoot BP Plus)对人牙髓干细胞(hDPSCs)增殖、分化为成牙本质细胞的促进作用。方法①不同浓度四种盖髓剂对hDPSCs增殖的促进作用观察。取...目的观察矿物三氧化物凝聚体(Mineral Trioxide Aggregate,MTA)和三种改良盖髓剂(nRoot、Vitapex、iRoot BP Plus)对人牙髓干细胞(hDPSCs)增殖、分化为成牙本质细胞的促进作用。方法①不同浓度四种盖髓剂对hDPSCs增殖的促进作用观察。取第5代hDPSCs细胞分为MTA组、iRoot BP Plus组、nRoot组、Vitapex组及空白组,MTA组、iRoot BP Plus组、nRoot组、Vitapex组分别加入不同浓度(0.02、0.2、1、2 mg/mL)的MTA、iRoot BP Plus、nRoot和Vitapex培养液,空白组加入含10%FBS的DMEM/F12培养液。分别于培养24、48 h时,采用CCK-8法测算各组细胞增殖活性。②四种盖髓剂对hDPSCs分化为成牙本质细胞的促进作用观察。通过ALP活性检测筛选出四种材料对hDPSCs最适诱导浓度。取第4代hDPSCs分为五组:空白组、MTA组、iRoot BP Plus组、nRoot组和Vitapex组换为0.2 mg/mL的MTA、iRoot BP Plus、nRoot和Vitapex成骨培养基,空白组为正常成骨培养基。培养第21天时采用茜素红染色和半定量分析法观察各组细胞矿化结节形成情况,培养第7天时采用Western Blotting法检测细胞相关蛋白类核转录因子(Runt-related transcription factor 2,RUNX-2)、骨钙素(Osteocalcin,OCN)、牙本质涎磷蛋白(Dentin sialophosphoprotein,DSPP)以及牙本质基质蛋白1(Dentin matrix protein 1,DMP-1)。结果与空白组相比,培养第2天时0.02、0.2、1 mg/mL的MTA组、iRoot BP Plus在和nRoot组细胞增殖活性高(P均<0.05);与培养第1天时相比,培养第2天时2 mg/mL的MTA组和Vitapex组细胞增殖活性低(P均<0.05)。筛选0.2 mg/mL为后续受试浓度。与nRoot组和Vitapex组相比,iRoot BP Plus组和MTA组细胞钙沉积量高(P均<0.05)。与空白组相比,MTA组、iRoot BP Plus组、nRoot组和Vitapex组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均升高(P均<0.05);与nRoot组、Vitapex组相比,iRoot BP Plus组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均高(P均<0.05)。结论相比于MTA、Vitapex,nRoot和iRoot BP Plus更能促进hDPSCs的细胞增殖、诱导细胞分化为成牙本质细胞。展开更多
基金supported by the National Natural Science Foundation of China (No. 82071110 and No. 82230029) to Zhi Chenthe National Natural Science Foundation of China (No. 82071077 and No.82270948)+1 种基金“the Fundamental Research Funds for the Central Universities”“The Young Top-notch Talent Cultivation Program of Hubei Province” to Huan Liu
文摘Mouse dental papilla cells(mDPCs)are cranial neural crest-derived dental mesenchymal cells that give rise to dentin-secreting odontoblasts after the bell stage during odontogenesis.The odontoblastic differentiation of mDPCs is spatiotemporally regulated by transcription factors(TFs).Our previous work reveals that chromatin accessibility was correlated with the occupation of the basic leucine zipper TF family during odontoblastic differentiation.However,the detailed mechanism by which TFs regulate the initiation of odontoblastic differentiation remains elusive.Here,we report that phosphorylation of ATF2(p-ATF2)is particularly increased during odontoblastic differentiation in vivo and in vitro.ATAC-seq and p-ATF2 CUT&Tag experiments further demonstrate a high correlation between p-ATF2 localization and increased chromatin accessibility of regions near mineralization-related genes.Knockdown of Atf2 inhibits the odontoblastic differentiation of mDPCs,while overexpression of p-ATF2 promotes odontoblastic differentiation.ATAC-seq after overexpression of p-ATF2 reveals that p-ATF2 increases the chromatin accessibility of regions adjacent to genes associated with matrix mineralization.Furthermore,we find that p-ATF2 physically interacts with and promotes H2BK12 acetylation.Taken together,our findings reveal a mechanism that p-ATF2 promotes odontoblastic differentiation at initiation via remodeling chromatin accessibility and emphasize the role of the phosphoswitch model of TFs in cell fate transitions.
文摘目的观察矿物三氧化物凝聚体(Mineral Trioxide Aggregate,MTA)和三种改良盖髓剂(nRoot、Vitapex、iRoot BP Plus)对人牙髓干细胞(hDPSCs)增殖、分化为成牙本质细胞的促进作用。方法①不同浓度四种盖髓剂对hDPSCs增殖的促进作用观察。取第5代hDPSCs细胞分为MTA组、iRoot BP Plus组、nRoot组、Vitapex组及空白组,MTA组、iRoot BP Plus组、nRoot组、Vitapex组分别加入不同浓度(0.02、0.2、1、2 mg/mL)的MTA、iRoot BP Plus、nRoot和Vitapex培养液,空白组加入含10%FBS的DMEM/F12培养液。分别于培养24、48 h时,采用CCK-8法测算各组细胞增殖活性。②四种盖髓剂对hDPSCs分化为成牙本质细胞的促进作用观察。通过ALP活性检测筛选出四种材料对hDPSCs最适诱导浓度。取第4代hDPSCs分为五组:空白组、MTA组、iRoot BP Plus组、nRoot组和Vitapex组换为0.2 mg/mL的MTA、iRoot BP Plus、nRoot和Vitapex成骨培养基,空白组为正常成骨培养基。培养第21天时采用茜素红染色和半定量分析法观察各组细胞矿化结节形成情况,培养第7天时采用Western Blotting法检测细胞相关蛋白类核转录因子(Runt-related transcription factor 2,RUNX-2)、骨钙素(Osteocalcin,OCN)、牙本质涎磷蛋白(Dentin sialophosphoprotein,DSPP)以及牙本质基质蛋白1(Dentin matrix protein 1,DMP-1)。结果与空白组相比,培养第2天时0.02、0.2、1 mg/mL的MTA组、iRoot BP Plus在和nRoot组细胞增殖活性高(P均<0.05);与培养第1天时相比,培养第2天时2 mg/mL的MTA组和Vitapex组细胞增殖活性低(P均<0.05)。筛选0.2 mg/mL为后续受试浓度。与nRoot组和Vitapex组相比,iRoot BP Plus组和MTA组细胞钙沉积量高(P均<0.05)。与空白组相比,MTA组、iRoot BP Plus组、nRoot组和Vitapex组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均升高(P均<0.05);与nRoot组、Vitapex组相比,iRoot BP Plus组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均高(P均<0.05)。结论相比于MTA、Vitapex,nRoot和iRoot BP Plus更能促进hDPSCs的细胞增殖、诱导细胞分化为成牙本质细胞。
