Chir99021调控Wnt/β-catenin信号通路干预干细胞成骨/成牙分化的研究

Chir99021 Regulates Osteogenic/Dentinogenic Differentiation of Dental Pulp Stem Cells through Wnt/β-catenin Signaling Pathway

目的:探讨Chir99021调控Wnt/β-catenin信号通路干预人牙髓干细胞成骨/成牙分化及其可能的机制。方法:分离纯化和培养人前磨牙牙髓干细胞(HDPSCs),免疫荧光检测牙本质涎蛋白(dentin sialoprotein,DSP)和波形蛋白(Vimentin),CCK-8检测不同浓度Chir99021对HDPSCs细胞增殖作用的影响。将HDPSCs分为3组培养,第1组正常培养,为空白对照组;第2组成骨诱导液培养,为实验对照组;第3组成骨诱导液+Chir99021培养,为实验组。15 d后用茜素红染色对各组细胞进行钙沉积物检测;qPCR于5、10、15 d检测各组轴抑制蛋白2(axisinhibition protein2,Axin2)、β连环蛋白(β-catenin)及糖原合成激酶3(GSK-3β)水平;Western blot于5、10、15 d检测各组RUNT相关转录因子2(Runx2)、碱性磷酸酶(ALP)、骨钙素(OC)、牙本质涎磷蛋白(DSPP)和Ⅰ型胶原的表达。结果:分离培养后的HDPSCs中DSP和Vimentin呈阳性表达;Chir99021浓度为2000 nmol/L时,细胞增殖情况最明显;茜红素染色显示实验组钙沉积物较其余两组显著增加;实验组中Axin2、β-catenin显著上升,GSK-3β显著下降(P<0.05);Runx2、ALP在实验组及实验对照组中有显著表达(P<0.05),OC在实验组中显著表达(P<0.05);DSPP、Ⅰ型胶原在实验组中显著表达(P<0.05)。结论:Chir99021通过激活Wnt/β-catenin信号通路促进HDPSCs的增殖和成骨/成牙分化。

Objective:To investigate the effect of osteogenic and dentiparous differentiation of human dental pulp stem cells through Wnt/β-catenin signaling pathway controlled by Chir99021 and its possible mechanism.Methods:HDPSCs were purified and cultured.DSP and Vimentin were detected by immunofluorescence.The effects of different concentrations of Chir99021 on HDPSCs proliferation was detected by CCK-8.Three groups were prepared according to the culture medium,as follows:blank control group was normal culture,experimental control group was osteogenic induction culture,and experimental group was osteogenic induction and Chir99021 culture.Lycopene staining method was used to detect calcium deposits.qPCR was used to detect Axin2,β-catenin,and GSK-3βin day 5,10,and 15.Western blotting was used to detect Runx2,ALP,OC,DSPP and collagenⅠin day 5,10,and 15.Results:DSP and Vimentin were positive in purified HDPSCs.The best concentration of Chir99021 was 2000 nmol/L.The calcium deposits of experimental group were significantly more than other groups.β-catenin and Axin2 of experimental group was significantly higher than those of other groups(P<0.05).GSK-3βof experimental group was significantly lower(P<0.05).Runx2 and ALP were significantly expressed in the control group and experimental group(P<0.05).OC of experimental group was significantly higher(P<0.05).DSPP and collagenⅠwere significantly expressed in the experimental group(P<0.05).Conclusion:Osteogenic and dentiparous differentiation of HDPSCs were activated by Wnt/β-catenin signaling pathway controlled by Chir99021.