Wnt/β-catenin信号通路在牙损伤修复中的作用

Effect of Wnt/β-catenin signaling pathway in tooth injury restoration

目的通过构建牙本质-牙髓损伤小鼠模型,探讨Wnt/β-catenin信号在其修复中的作用。方法选择雌性昆明小鼠40只,鼠龄6~8周,体质量18~22 g。将其随机分为Control组、Model组、LiCl组和DKK1组,每组10只。Control组常规饲养至实验结束,Model组、LiCl组和DKK1组均建立牙本质-牙髓损伤模型并通过牙周膜分别注射0.9%氯化钠溶液(生理盐水)、LiCl(激活Wnt/β-catenin信号通路)和DKK1(抑制Wnt/β-catenin信号通路)干预。损伤后7 d处死小鼠,通过苏木精-伊红(HE)染色,从形态学上观察牙损伤后的情况;Western blot和实时定量聚合酶链式反应(PCR)分别检测Wnt10a、胞内-β连锁蛋白(β-catenin)、支架蛋白Axin2及牙本质涎磷蛋白(DSPP)的mRNA和蛋白水平改变。结果实验成功构建了牙本质-牙髓损伤小鼠模型。使用LiCl和DKK1进行干预,HE染色结果显示LiCl组损伤部位得到良好的修复,Model组的牙本质、牙髓损伤修复不明显,DKK1组的损伤部位则几乎未修复。Western blot和实时定量PCR结果显示,Control组与Model组相比,Wnt10a mRNA、β-catenin mRNA、DSPP mRNA和Axin2 mRNA表达水均下降(Control组:1.003±0.096、1.003±0.100、1.014±0.200、1.000±0.033;Model组:1.724±0.172、1.971±0.261、2.090±0.116、1.827±0.066),且β-catenin mRNA和Axin2 mRNA下降显著(P<0.05);Li Cl组与Model相比,Wnt10a mRNA、β-catenin mRNA、DSPP mRNA和Axin2 mRNA表达水平均上升(LiCl组:3.497±0.475、2.463±0.242、3.563±0.181、2.576±0.326),而DKK1组均下降(1.096±0.141、1.220±0.245、1.163±0.272、1.169±0.047),差异均有统计学意义(P<0.05)。与Control组相比,Model组Wnt10a、β-catenin、DSPP和Axin2蛋白表达水平均下降(Model组:0.511±0.030、0.565±0.029、0.461±0.049、0.315±0.049;Control组:0.609±0.036、0.834±0.038、0.663±0.013、0.480±0.174),差异均有统计学意义(P<0.05);与Model组相比,Wnt10a、β-catenin、DSPP和Axin2蛋白表达水平在LiCl组上升(0.719±0.041、0.740±0.097、0.557±0.016、0.712±0.092),在DKK1组下降(0.418±0.035、0.019±0.010、0.138±0.017、0.453±0.013),差异均有统计学意义(P<0.05)。结论在牙本质-牙髓损伤中激活Wnt/β-catenin通路可加快牙本质形成,促进牙损伤的修复。

Objective To explore effect of Wnt/β-catenin signaling pathway in restoration of dentin-pulp injury by constructing mouse model.Methods Forty female Kunming mice were sacrificed,which aged 6-8 weeks with body mass of 18-22 g.All of them were randomly divided into Control group,Model group,LiCl group and Dickkopf 1(DKK1)group,10 in each group.The Control group was routinely fed until experiment terminal,The Model group,Li Cl group and DKK1 group constructed dentin-pulp injury models and injected 0.9%sodium chloride solution(physiological saline),LiCl(activating Wnt/β-catenin signaling pathway)and DKK1(inhibiting Wnt/β-catenin signaling pathway).Mice were sacrificed 7-day after injury,and the condition after tooth injury was observed morphologically by hematoxylin-eosin(HE)staining;The Western blot and re al-time quantitative polymerase chain reaction(PCR)were used to detect change of Wnt10a,intracellular-β-catenin(β-catenin),scaffold protein Axin2 and dentin sialophosphoprotein(DSPP)mRNA and protein levels.Results The mouse model of dentinpulp injury was successfully constructed.LiCl and DKK1 were intervened,the results of HE staining showed that injury of LiCl group was well restored,the injury restoration of dentin-pulp in model group was unobvious,and injury of DKK1 group was almost unrestored.The Western blot and real-time quantitative PCR results showed that compared with Model group,the expression levels of Wnt10a mRNA,β-catenin mRNA,DSPP mRNA and Axin2 mRNA decreased in Control group(Control group:1.003±0.096,1.003±0.100,1.014±0.200,1.000±0.033;Model group:1.724±0.172,1.971±0.261,2.090±0.116,1.827±0.066),whileβ-catenin mRNA and Axin2 mRNA decreased significantly(P<0.05).Compared with Model group,the expression of Wnt10a mRNA,β-catenin mRNA,DSPP mRNA and Axin2 mRNA increased in Li Cl group(Li Cl group:3.497±0.475,2.463±0.242,3.563±0.181,2.576±0.326),while DKK1 group were deceased(1.096±0.141,1.220±0.245,1.163±0.272,1.169±0.047),and the differences were statistical significance(P<0.05).Compared with Control group,the protein expression levels of Wnt10a,β-catenin,DSPP and Axin2 were decreased in Model group(Model group:0.511±0.030,0.565±0.029,0.461±0.049,0.315±0.049;Control group:0.609±0.036,0.834±0.038,0.663±0.013,0.480±0.174),and the differences were statistical significance(P<0.05).Compared with Model group,the expression of Wnt10a,β-catenin,DSPP and Axin2 were increased in Li Cl group(0.719±0.041,0.740±0.097,0.557±0.016,0.712±0.092),and decreased in DKK1 group(0.418±0.035,0.019±0.010,0.138±0.017,0.453±0.013),and the differences were statistical significance(P<0.05).Conclusion It is demonstrated that Wnt/β-catenin pathway could regulate the expression of DSPP,which accelerate dentin formation and promote restoration of tooth injury.