【作者】 杜宇； 凌均棨；

【Author】 Du Yu,Ling Jun-qi~*(Department of Conservative Dentistry and Endodontics,Guanghua School of Stomatology,Sun Yat-sen University,Guangzhou 510055,China)

【机构】 中山大学光华口腔医学院牙体牙髓科；

【摘要】 目的:Wnt/β-catenin经典信号通路和干细胞生理功能密切相关,相关研究发现牙囊中含有多向分化潜能的干细胞,然而有关Wnt/β-catenin通路在牙囊细胞中的作用尚不清楚,本研究旨在探讨β-catenin在牙囊细胞中的表达及作用。方法:从出生6～7天的大鼠下颌骨分离培养牙囊细胞,鉴定成骨及成脂向分化能力。用Wnt/β-catenin通路活化剂LiCI刺激牙囊细胞,免疫荧光观察β-catenin定位,CCK8检测细胞增殖,并将重组DKK1作为Wnt/β-catenin通路抑制剂观察其对抗LiCI作用。此外,对1、3、5、7、9、1 1和13天的大鼠牙囊组织分别采用免疫组化检测β-catenin在体内的表达。结果:贴壁方法可成功获得大鼠牙囊细胞,具有成骨及成脂向分化能力。免疫荧光实验表明,未受LiCI刺激时β-catenin在牙囊细胞胞浆表达,LiCI刺激6h到24h时β-catenin转移至胞核,DKK1可有效抑制转核作用。CCK8显示10mmol/L的LiCI刺激24h促进牙囊细胞增殖,该效应可被DKK1抑制。免疫组化显示β-catenin在1天大鼠牙囊无表达,从3天到13天逐步提高。结论:大鼠牙囊细胞具有和其它牙源性干细胞类似的多向分化潜能。LiCI可使β-catenin从胞浆转移至胞核并促进细胞增殖,且能被DKK1拮抗。牙囊中β-catenin表达提高表明其可能和细胞分化相关,提示Wnt/β-catenin信号通路可能在牙囊细胞中发挥重要作用。更多还原

【Abstract】 Objective:Canonical Wnt/B -catenin signaling pathway plays significant role in stem cell fate determination.Dental follicle cells(DFC) have been reported to contain stem cells with multiple-directional differentiation potential.However,the function of Wnt/B -catenin pathway in DFC remains unclear.The aim of this study was to detect the expression of B -catenin and its possible roles in DFC.Materials and Methods:DFC were cultured from mandibles of postnatal rats on day 6 to 7,as well as examined by their osteogenesis and adipogenesis abilities.LiCI was employed here as a Wnt/β-catenin pathway activator to stimulate DFC in vitro followed by immunofluorescence and CCK8 proliferation assay.Moreover,recombinant Dickkopf-related protein 1(DKK1) was also applied as an antagonist of Wnt/β-catenin pathway to inhibit the effect of LiCI.In vivo, immunohistochemistry was performed on dental follicles of postnatal rats from day 1,3, 5,7,9,11 and 13.Results:DFC can be successfully obtained by plastic adherence and exhibited effective osteogenesis and adipogenesis abilities.In vitro,immunofluorescence assay showedβ-catenin was located in cytoplasm of DFC without LiCI treatment. Nuclear(3 -catenin staining was detected after LiCI stimulation from 6h to 24h.,while DKK1 could effectively antagonist the nuclear transfer of LiCI on DFC.CCK8 results indicated LiCI at a concentration of 10mmol/L may promote the proliferation of DFC after 24h,and it could be suppressed by DKK1.Immunohistochemistry results demonstratedβ-catenin was absent on day 1 and increased gradually from day 3 to 13 on rat dental follicles.Conclusions:Cultured rat DFC showed similar multiple-directional differentiation potential to other dental stem cells.LiCI stimulation was capable to evokeβ-catenin from cytoplasm to nucleus and promote the proliferation of DFC,however inhibited by DKK1.β-catenin located in dental follicles increased chronologically,which may be related to differentiation of DFC,suggesting an important role of Wnt/β-catenin signaling pathway in cell function of DFC.更多还原