Wnt3a蛋白对牙髓干细胞成骨向分化的影响

Effects of Wnt3a on osteogenic differentiation of dental pulp stem cells

目的探讨Wnt3a蛋白对牙髓干细胞（dentalpulpstemcells，DPSC）成骨向分化的影响，进一步说明Wnt信号通路在DPSC分化中的作用。方法将不同质量浓度『0（培养液组）、5、20、50及100vg／L1的Wnt3a蛋白作用于DPSC，于诱导培养的第7天检测其碱性磷酸酶（alkalinephosphatase，ALP）活性，对检测结果进行单因素方差分析；茜素红染色检测矿化结节形成情况；通过实时荧光定量PCR（quantitativereal．timePCR，qPCR）检测骨涎蛋白、I型胶原、Runt相关转录因子2（Runt—relatedtranscriptionfactor-2，RUNX2）及骨钙蛋白4种骨源性基因的表达，对结果数据采用独立样本t检验进行统计学分析。结果DPSC诱导7d后检测结果显示，Wnt3a蛋白可抑制ALP的活性，0、5、20、50及100μg/L组ALP的活性分别为1．076±0．203、0．828±0．118、0．505±0．044、0．499±0．038及0．483±0．060，Wnt3a蛋白质量浓度越高对ALP活性的抑制作用越强；qPCR检测显示，5txg／LWnt3a蛋白组骨钙蛋白基因阳性表达水平（0．092±0．005）显著低于培养液组（0．858±0．190）（P〈0．05）；28d后茜素红染色结果显示，5μg／LWnt3a蛋白对DPSC无明显的诱导矿化现象。结论Wnt3a蛋白可抑制DPSC成骨向分化。

Objective To investigate the effect of Wnt3a on osteogenic differentiation of human dental pulp stem cells （DPSC）. Methods DPSCs were subjected to different concentrations of Wnt3a （0, 5, 20, 50 and 100 μg/L） and at seven days after culture the alkaline phosphatase （ALP） activity was tested. Mineralized nodule formation was examined by alizarin red staining. Osteogenic-related gene expression of bone sialoprotein （BSP）, osteocalcin （OCN）, collagen type I （COL- I ）, Runt-related transcription factor-2 （RUNX2） was examined by quantitative real-time PCR （qPCR）. Results After seven days of induction by DPSC, Wnt3a protein could inhibit the ALP activity （concentration 0： 1.076±0.203, 5 μg/L： 0.828±0.118, 20 μg/L： 0.505±0.044, 50 μg/L： 0.499±0.038, 100 μg/L： 0.483±0.060）. The expression of OCN in 5 μg/L Wnt3a group （0,092±0.005） was lower than that in culture medium （0.858±0.190）（P〈0.05）. Alizarin red staining showed that 5 μg/L Wnt3a had no mineralization induction effect on DPSC. Conclusions Wnt3a could inhibit osteogenic differentiation of dental pulp stem cells.