整合素α5β1在苯妥英钠促进大鼠PDLSCs和BMMSCs黏附于牙骨质中的作用

The role of integrin α5β1 in phenytoin promoting the adhesion of rPDLSCs and rBMMSCs to cementum in rat

目的探讨在苯妥英钠(Phenytoin,PHT)促进大鼠牙周膜干细胞(Rat periodontal ligament stem cells,rPDLSCs)、大鼠骨髓间充质干细胞(Rat Bone Marrow Mesenchymal Stem Cells,rBMMSCs)黏附于牙骨质过程中,整合素α5β1(Integrinα5β1)起到的作用。方法提取大鼠BMMSCs和PDLSCs,培养并纯化。通过细胞鉴定后,将获得的两种细胞各分为4组:40 mg/L PHT处理组、40 mg/L PHT+整合素α5抗体处理组、40 mg/L PHT+整合素β1抗体处理组、PBS处理组,每组细胞放入置有牙骨质片的96孔板处理4 h后,检测黏附于牙骨质片上的细胞量并做以比较。最后,利用qRT-PCR和Western blot检测40 mg/L PHT组与对照组细胞的整合素α5、β1亚基的mRNA与蛋白表达量。结果40 mg/L PHT可促进rBMMSCs及rPDLSCs黏附于牙骨质片,加入整合素α5、β1抗体后,均明显抑制了40 mg/L PHT对rBMMSCs、rPDLSCs黏附于牙骨质的促进作用(P<0.01)。qRT-PCR、Western-blot结果显示PHT处理组的整合素α5、β1亚基表达量高于空白对照组(P<0.05)。结论40 mg/L PHT能促进rBMMSCs、rPDLSCs黏附于牙骨质,该作用与整合素α5β1的表达上调密切相关。

Objective To explore the effects of Phenytoin(PHT)on attachment of rat periodontal ligament stem cells(rPDLSCs)and rat bone marrow mesenchymal stem cells(rBMMSCs)to cementum,and the role of integrin α5β1 in this process.Methods The rPDLSCs and rBMMSCs were isolated and purified.After cell identification,the cells obtained were divided into 4 groups:40 mg/L PHT treatment group,40 mg/L PHT+integrinα5 antibody,40 mg/L PHT+integrinβ1 antibody group,PBS treatment group.When cells were co-cultured with cementum chips for 4 hours,the amount of cells attached to the cementum chips in different groups was tested and compared.Cells in every groups are detected for integrin α5,β1 mRNA and protein expression levels by using real-time quantification PCR,Western blot.Results 40 mg/L PHT can promote the attachment of cells.After adding anti-integrin α5,β1 antibodies,the promotion of the adhesion of rBMMSCs and rPDLSCs to cementum by 40 mg/L PHT was significantly inhibited(P<0.01).The results of real-time quantification PCR,Western-blot both showed that the expression level of integrinα5β1 in experimental group is higher than that in control group(P<0.05).Conclusion 40 mg/L PHT can promote rBMMSCs and rPDLSCs attach to cementum chips’surface,this effect is related to the up-regulation of integrinα5β1.