PDGFD对人牙髓干细胞迁移及成牙本质向分化的作用

Effects of platelet-derived growth factor D on migration and odontogenic differentiation of human dental pulp stem cells

目的:探讨人重组血小板来源的生长因子D(platelet-derived growth factor D,PDGFD)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)迁移及成牙本质向分化的作用。方法:利用酶解法分离培养hDPSCs,流式细胞术鉴定所培养的间充质干细胞表面分子标志物的表达,诱导hDPSCs三系分化并使用相应染色鉴定,以表征其多向分化潜能。应用细胞划痕实验检测PDGFD对hDPSCs迁移能力的影响,利用实时荧光定量PCR及蛋白免疫印迹法检测PDGFD对hDPSCs成牙本质相关mRNA及蛋白表达的影响,利用碱性磷酸酶(alkaline phosphataseI,ALP)和茜素红(alizarin red staining,ARS)染色检测PDGFD对hDPSCs矿化的影响。采用SPSS 26.0软件包对数据进行统计学分析。结果:细胞形态学分析、流式细胞术鉴定和三系分化结果显示,所分离得到的细胞符合hDPSCs特征,并且具有多向分化潜能。细胞划痕实验结果表明,12 h时,仅50 ng/mL的PDGFD对hDPSCs的迁移能力有影响;24 h时,10和50 ng/mL的PDGFD对hDPSCs的迁移能力均有影响。PCR结果显示,10与50 ng/mL的PDGFD均对hDPSCs的成牙本质分化有促进作用,50 ng/mL的PDGFD对hDPSCs的成牙本质分化更为显著。蛋白免疫印迹实验、ALP及ARS染色所得结果和PCR结果相同。结论:成功分离并培养了具有典型间充质干细胞表型和多向分化潜能的hDPSCs。10和50 ng/mL浓度的PDGFD对hDPSCs的迁移和成牙本质向分化均有促进作用,其中50 ng/mL的PDGFD的促进作用更为显著。

PURPOSE:To investigate the effect of human recombinant platelet-derived growth factor D(PDGFD)on migration and odontogenic differentiation of human dental pulp stem cells(hDPSCs).METHODS:Primary hDPSCs were isolated by enzymolysis and cultured.The expression of molecular markers on the surface of cultured mesenchymal stem cells was identified by flow cytometry.Three lines of hDPSCs were induced and identified by corresponding staining to characterize their potential for multidirectional differentiation.The effect of PDGFD on the migration ability of hDPSCs was investigated by cell scratching test.RT-PCR and Western blotting were used to detect the effects of PDGFD on the expression of odontoblast-related mRNA and proteins in hDPSCs.Alkaline phosphatase(ALP)and alizarin red staining(ARS)were used to detect the effect of PDGFD on the mineralization of hDPSCs.SPSS 26.0 software package was used for data analysis.RESULTS:Morphological analysis,flow cytometry identification and three-line differentiation showed that the isolated cells were consistent with the characteristics of hDPSCs and had multidirectional differentiation potentials.The results of cell scratching test showed that only 50 ng/mL PDGFD at 12 h had an effect on the migration ability of hDPSCs,but both 10 and 50 ng/mL PDGFD at 24 h had an effect on the migration ability of hDPSCs.PCR results showed that PDGFD of 10 and 50 ng/mL could promote the odontogenic differentiation of hDPSCs,and PDGFD of 50 ng/mL had a more significant effect on the odontogenic differentiation of hDPSCs.The results of Western blotting,ALP and ARS were the same as those of PCR.CONCLUSIONS:hDPSCs with typical mesenchymal stem cell phenotype and multidirectional differentiation potential were successfully isolated and cultured.PDGFD at the concentration of 10 ng/mL and 50 ng/mL can promote the migration and odontogenic differentiation of human dental pulp stem cells,and the promotion effect of PDGFD at the concentration of 50 ng/mL is more significant.