摘要
目的探讨Chir99021对大鼠牙髓干细胞(DPSCs)成骨诱导分化的影响及机制。方法原代分离大鼠DPSCs,利用荧光免疫法进行验证。设置不同浓度的Chir99021,通过CCK-8检测细胞增殖情况挑选最适浓度。利用茜素红染色验证细胞成骨诱导分化。最后通过Real-time PCR、Western blotting检测成骨诱导分化相关基因与蛋白无翅型MMTV整合位点家族成员1(Wnt1)、无翅型MMTV整合位点家族成员3A(Wnt3a)、β-连环蛋白(β-catenin)、轴抑制蛋白2抗体(Axin2)、骨钙素(OCN)、牙本质基质蛋白1(DMP1)的mRNA和蛋白表达情况。结果牙本质涎磷蛋白(DSPP)和干细胞标记物波形蛋白(vimentin)阳性表达,说明大鼠DPSCs分离成功。大鼠DPSCs成骨诱导后钙沉积物显著上升,加入糖原合成酶激酶-3β(GSK-3β)的小分子抑制剂Chir99021后钙沉积物明显减少。大鼠DPSCs成骨诱导分化后Wnt1、Wnt3a、β-catenin、Axin2、OCN和DMP1表达明显上升,而加入Chir99021后Wnt1、Wnt3a、β-catenin、Axin2、OCN和DMP1蛋白表达均显著下降。结论Chir99021对诱导7 d后的大鼠DPSCs成骨诱导分化有一定的抑制作用。
Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells.Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay.Different concentrations of Chir99021 were set,and the cell proliferation was detected by CCK-8 to select the optimal concentration.Osteogenic differentiation was detected by alizarin red staining.The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1(Wnt1),Wnt3a and Wnt3aβ-expression of catenin,axis inhibition protein 2(Axin 2),dentin sialophosphoprotein(OCN)and dentin matrix acidic phosphoprotein 1(DMP1)was detected by Real-time PCR and Western blotting.Results The positive expression of dentin sialophosphoprotein(DSPP)and vimentin indicated that rat dental pulp stem cells were successfully isolated.After osteogenic induction of rat dental pulp stem cells,calcium deposits significantly increased with the addition of glycogen synthase kinase-3β(GSK-3β)inhibitor Chir99021,calcium deposits were significanted reduced.After osteogenic differentiation of rat dental pulp stem cells,the expression of Wnt1,Wnt3a,β-catenin,Axin2,OCN and DMP1 increased,while the expression of Wnt1,Axin2,OCN and DMP1 decreased with the addition of Chir99021.Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.
作者
谢惠兰
方芳
林毅
XIE Hui-lan;FANG Fang;LIN Yi(Department of Stomatology,Fujian Provincial Hospital,Fuzhou 350001,China;Provincial Clinical Medical College of Fujian Medical University,Fuzhou 350001,China)
出处
《解剖学报》
CAS
CSCD
2024年第1期67-72,共6页
Acta Anatomica Sinica
基金
福建省自然科学基金(2019J01503)。
