无血清培养下富血小板纤维蛋白提取液对牙髓干细胞增殖分化的影响

Effect of Platelet-rich Fibrin Extract on Proliferation and Differentiation of Dental Pulp Stem Cells in Serum-free Culture

目的:在无血清培养条件下,使用富血小板纤维蛋白提取液(platelet-rich fibrin extract,PRFe)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)增殖及分化的影响。方法:从健康志愿者的阻生齿中分离培养hDPSCs,经细胞形态和流式细胞技术对其鉴定。实验组中使用含PRFe不含胎牛血清(fetal bovine serum,FBS)的α-最低必须培养基(α-minimal essential medium,α-MEM),对照组中使用含10%FBS的α-MEM。噻唑蓝(methyl thiazol tetrazolium,MTT)法检测培养1~7d的细胞增殖情况。成骨能力的测定采用碱性磷酸酶染色,实时荧光定量聚合酶链式反应(real time quantitive-polymerase chain reaction,RT-PCR)检测碱性磷酸酶(alkaline phosphatase,ALP)、骨钙蛋白(osteocalcin,OCN)、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)和runt相关转录因子2(runt-related transcription factor-2,RUNX-2)的表达。结果:hDPSCs高表达CD44、CD90和CD105,低表达CD34和CD45。两种培养基培养细胞形态相似,实验组的细胞形态更为纤长。随着诱导时间的增加,两组的成骨分化能力均上调(P<0.05),实验组成骨分化能力强于对照组(P<0.05)。结论:适宜浓度PRFe能够维持hDPSCs增殖分化。

Objective:To culture human dental pulp stem cells(hDPSCs)in vitro by using platelet-rich fibrin extract(PRFe)with serum-free medium,and analyze their proliferation and differentiation.Methods:hDPSCs were isolated from impacted third molars of young healthy donors and identified by stem cell surface marker expression using flow cytometry.Experimental group usedα-minimal essential medium containing PRFe without fetal bovine serum,while control group used theα-MEM with 10%FBS.The two groups were analyzed for:morphology,growth characteristics,alkaline phosphatase activity using ALP staining,and the gene expression of BMP2,RUNX-2,OCN,and ALP using real-time PCR.Results:hDPSCs had high expression of markers CD44,CD90,CD105,and low expression of markers CD34 and CD45.hDPSCs cultured in the experimental group showed a similar morphology as cultured in the control group but more thin,long and stereoscopic.Under osteogenesis induction,the osteogenic differentiation ability of two groups significantly increased(P<0.05).However,the expression of experimental group was higher than that of control group(P<0.05).Conclusion:PRFe can support the proliferation and osteogenic differentiation of hDPSCs in vitro.