脐带血清促进牙髓干细胞增殖作用研究

Effect of umbilical cord sera on dental pulp stem cell proliferation

目的建立脐带血清(CBS)培养体系,替代胎牛血清(FBS)体外扩增牙髓干细胞(DPSC)。方法取各项病原微生物测定皆显示阴性的健康人CBS用作细胞培养。提取健康成人完整且无龋坏的正畸拔除牙或第三磨牙的DPSC,设置四个实验组,其中对照组为含10%FBS培养基培养的DPSC,其他三组分别为含5%、10%、20%CBS培养基培养的DPSC。经由MTT法对比四组细胞增殖速度;对增殖速度最快的一组,通过不同诱导液对其DPSC施以诱导,使其分别分化为脂肪细胞、成骨细胞、成软骨细胞,对其多向分化活性加以证实;流式细胞术(FCM)测定细胞表面标记物表达。结果脐带血清可培养出能够稳定传代的DPSC;MTT结果显示,各组牙髓干细胞在1、3、5、7、9 d内均呈现不同程度的生长状态,且呈线性增长,呈“S”型。其中10%CBS组在第7 d后细胞增殖较为明显;培养出的DPSC具有多向分化能力;FCM测定培养出的第3代DPSC表面标记物表达水平,MSCA-1、CD73与CD90为阳性表达,CD34与CD45为阴性表达,与间充质干细胞表面标记物表达特性相符。结论CBS培养体系于体外条件下对DPSC增殖具促进作用,且培养所得DPSC具备干细胞特性。

Objective To investigate the possibility of the cord blood serum(CBS)culture to replace fetal bovine serum(FBS)for in vitro expansion of dental pulp stem cells(DPSC).Methods Healthy human CBS that were negative for pathogenic microorganisms were utilized for culture of DPSC,which were obtained from orthodontically extracted teeth or third molars in healthy adults without caries.Four experimental groups were set,i.e.,DPSC culture with 10%FBS-containing medium as the control,while the other three DPSC groups were cultured with 5%,10%,and 20%CBS medium,respectively.Cell viability MTT assay was used to assess changed cell viability in vitro.After that,the fastest proliferating group of DPSCs was then induced with different induction solutions to differentiate them into adipocytes,osteoblasts and chondrocytes to confirm their multidirectional differentiation capacity.Expression of the cell surface markers was determined by using the flow cytometry(FCM).Results Umbilical cord serum addition was able to grow DPSCs and produce DPSC capable of stable transmission.The MTT results showed that all groups of dental pulp stem cells present different degrees of growth rates at 1,3,5,7 and 9 days with the linear and"S"shaped growth curves.Moreover,DPSCs grown in the 10%CBS-containing medium showed more obvious growth advantage after the 7 day of culture and these cultivated DPSC possessed multidirectional differentiation capacities.The surface markers(MSCA-1,CD73,and CD90)were expressed,whereas CD34 and CD45 were not expressed in cultured 3rd generation DPSCs,which are consistent with the surface marker expression characteristics of MSCs.Conclusion Under the in vitro conditions,the CBS culture system was able to promote DPSC growth and multidirectional differentiation.