Human dental pulp stem cells(DPSCs)have emerged as an important source of stem cells in the tissue engineering,and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration...Human dental pulp stem cells(DPSCs)have emerged as an important source of stem cells in the tissue engineering,and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration.Nevertheless,little is known about the complicated molecular mechanisms.In this study,we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition.Hypoxia was induced in DPSCs by Cobalt chloride(CoCl_(2))treatment.The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase(ALP)activity,Alizarin Red S staining and main osteo/dentinogenic markers.A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p.This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment.Furthermore,miR-140-3p rescued the CoCl_(2)-induced decreased osteo/odontogenic differentiation potentials in DPSCs.Besides,we investigated that miR-140-3p directly targeted lysine methyltransferase 5B(KMT5B).Surprisingly,we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions.In conclusion,our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia,and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.展开更多
目的:评估iRoot BP Plus和ProRoot MTA作为盖髓剂对脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED)和人牙髓干细胞(human dental pulp stem cells,DPSC)生物学行为的影响.方法:选择处于生理性牙根吸收期...目的:评估iRoot BP Plus和ProRoot MTA作为盖髓剂对脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED)和人牙髓干细胞(human dental pulp stem cells,DPSC)生物学行为的影响.方法:选择处于生理性牙根吸收期的乳牙和无龋的正畸减数前磨牙或第三磨牙,体外提取、分离和培养SHED和DPSC,制备iRoot BP Plus和ProRoot MTA标准膜片并提取浸提液,通过CCK-8法检测l、3、5、7d时iRoot BP Plus和MTA浸提液对SHED和DPSC增殖能力的影响;Transwell小室和划痕修复实验观察iRoot BP Plus和MTA浸提液对SHED和DPSC迁移能力的影响;SHED和DPSC分别接种于iRoot BP Plus和MTA样品表面进行培养,分别在1、3、5d使用鬼笔环肽(phalloidin)和DAPI(4',6-diamidino-2-phenylindole)进行免疫荧光染色,观察细胞骨架变化;SHED和DPSC分别在成骨诱导培养基、含MTA浸提液的成骨诱导培养基和含iRoot BP Plus浸提液的成骨诱导培养基中进行矿化诱导,7d和14d时进行碱性磷酸酶(alkaline phosphatase,ALP)染色以及ALP定量分析,21 d时进行茜素红染色和半定量分析钙盐沉积量,采用SPSS 19.0软件包对数据进行统计学分析.结果:iRoot BP Plus和MTA均能促进SHED和DPSC的细胞增殖;细胞迁移与黏附实验中,iRoot BP Plus和MTA均能促进SHED和DPSC的迁移与黏附,其中,iRoot BP Plus的作用更显著(p=0.000);经矿化诱导后,iRoot BP Plus组的SHED和DPSC的ALP活性强于MTA组,茜素红染色和半定量分析钙盐沉积量结果显示,iRoot BP Plus和MTA均能促进细胞矿化,且iRoot BP Plus促进细胞矿化的能力显著强于MTA (P=0.000).结论:iRoot BP Plus和MTA均具有较好的生物相容性,均能促进SHED和DPSC的细胞增殖,能够促进细胞黏附、迁移,具有较好的成骨分化能力;且iRoot BP Plus促进SHED和DPSC黏附、迁移和成骨分化的能力相对MTA更好,iRoot BP Plus和MTA均可作为乳牙和年轻恒牙牙髓治疗的盖髓剂.展开更多
基金This work was supported by grants from the National Natural Science Foundation of China(81625005 to Z.P.F.)CAMS Innovation Fund for Medical Sciences(2019-I2M-5-031 to Z.P.F.)+1 种基金the Program for“Hundred-Thousand-Ten Thousand”Talents in Beijing(2018A16 to Z.P.F.)Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction(KFKT2019012 to L.L).
文摘Human dental pulp stem cells(DPSCs)have emerged as an important source of stem cells in the tissue engineering,and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration.Nevertheless,little is known about the complicated molecular mechanisms.In this study,we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition.Hypoxia was induced in DPSCs by Cobalt chloride(CoCl_(2))treatment.The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase(ALP)activity,Alizarin Red S staining and main osteo/dentinogenic markers.A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p.This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment.Furthermore,miR-140-3p rescued the CoCl_(2)-induced decreased osteo/odontogenic differentiation potentials in DPSCs.Besides,we investigated that miR-140-3p directly targeted lysine methyltransferase 5B(KMT5B).Surprisingly,we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions.In conclusion,our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia,and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.
文摘目的:评估iRoot BP Plus和ProRoot MTA作为盖髓剂对脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED)和人牙髓干细胞(human dental pulp stem cells,DPSC)生物学行为的影响.方法:选择处于生理性牙根吸收期的乳牙和无龋的正畸减数前磨牙或第三磨牙,体外提取、分离和培养SHED和DPSC,制备iRoot BP Plus和ProRoot MTA标准膜片并提取浸提液,通过CCK-8法检测l、3、5、7d时iRoot BP Plus和MTA浸提液对SHED和DPSC增殖能力的影响;Transwell小室和划痕修复实验观察iRoot BP Plus和MTA浸提液对SHED和DPSC迁移能力的影响;SHED和DPSC分别接种于iRoot BP Plus和MTA样品表面进行培养,分别在1、3、5d使用鬼笔环肽(phalloidin)和DAPI(4',6-diamidino-2-phenylindole)进行免疫荧光染色,观察细胞骨架变化;SHED和DPSC分别在成骨诱导培养基、含MTA浸提液的成骨诱导培养基和含iRoot BP Plus浸提液的成骨诱导培养基中进行矿化诱导,7d和14d时进行碱性磷酸酶(alkaline phosphatase,ALP)染色以及ALP定量分析,21 d时进行茜素红染色和半定量分析钙盐沉积量,采用SPSS 19.0软件包对数据进行统计学分析.结果:iRoot BP Plus和MTA均能促进SHED和DPSC的细胞增殖;细胞迁移与黏附实验中,iRoot BP Plus和MTA均能促进SHED和DPSC的迁移与黏附,其中,iRoot BP Plus的作用更显著(p=0.000);经矿化诱导后,iRoot BP Plus组的SHED和DPSC的ALP活性强于MTA组,茜素红染色和半定量分析钙盐沉积量结果显示,iRoot BP Plus和MTA均能促进细胞矿化,且iRoot BP Plus促进细胞矿化的能力显著强于MTA (P=0.000).结论:iRoot BP Plus和MTA均具有较好的生物相容性,均能促进SHED和DPSC的细胞增殖,能够促进细胞黏附、迁移,具有较好的成骨分化能力;且iRoot BP Plus促进SHED和DPSC黏附、迁移和成骨分化的能力相对MTA更好,iRoot BP Plus和MTA均可作为乳牙和年轻恒牙牙髓治疗的盖髓剂.
