富血小板纤维蛋白对牙髓干细胞体外增殖与分化特性的影响

Effect of platelet-rich fibrin(PRF) on the proliferation and differentiation of DPSCs in vitro

目的:探讨不同浓度富血小板纤维蛋白(platelet-rich fibrin,PRF)对犬牙髓干细胞(dental pulp stem cells,DPSCs)体外增殖及成牙本质/成骨分化能力的影响。方法:酶消法分离培养犬DPSCs,经流式细胞术和多向分化鉴定后分别在含自体PRF体积比为1/8、2/8、3/8的3种浓度的培养基中进行培养;采用MTT法检测1、2、3、4、5、6、7 d各时间点的细胞增殖活性;实时定量PCR检测培养7、14、21 d时ALP、DSPP、DMP1 mRNA的表达水平。结果:成功分离并获得克隆化培养的犬DPSCs,DPSCs具有成骨和成脂分化能力;3种浓度PRF均能促进DPSCs的增殖,1周内促增殖作用有时间依赖性而无PRF浓度依赖性;不同浓度PRF可通过上调成牙本质/成骨早期标志基因ALP、DSPP mRNA及晚期标志基因DMP1mRNA的表达来促进犬DPSCs成牙本质/成骨向分化,该效应既具时间依赖性又具有PRF浓度依赖性。结论:自体PRF可以不同方式同时促进犬DPSCs的增殖及分化。

AIM： To explore the biological effects of platelet-rich fibrin （PRF） on the proliferation and odontogenic/osteogenic differentiation of canine dental pulp stem cells （DPSCs） in vitro. METHODS ： DPSCs were obtained from primary culture of canine pulp cells and identified by cell surface markers with cytometric analysis. Mul tiple differentiation potential of DPSCs was explored by osteogenic and adipogenic inducing medium culture respectively followed by specific staining. PRF was obtained from the same dog and DPSCs were cultured in α-MEM with PRF at 1/8, 2/8 and 3/8 respectively. MTr assay was used to evaluate cell proliferation and real time PCR was performed to analyze the mRNA expression of ALP, DSPP and DMP1. RESULTS： Single cell-derived colony cultures were suc cessfully obtained and the osteogenic and adipogenic differentiation potential of DPSCs was confirmed by alizarin red staining and oil red staining. MTT assay demonstrated that PRF stimulated the proliferation of DPSCs in 7-day incuba tion in a time-dependent （P 〈 0.05） but not dose-dependent way （P 〉 0.05）. Real time PCR revealed that PRF pro moted the the expression of ALP, DSPP and DMP1 in a time-and dose-dependent way （P 〈 0.05）. CONCLUSION ： PRF can stimulate the proliferation and differentiation of DPSCs.