肌切蛋白adseverin对小鼠成牙本质细胞增殖、迁移及矿化作用的影响

Effect of adseverin on proliferation,migration and mineralization of MDPC-23 cells

目的通过构建沉默肌切蛋白(adseverin)的成牙本质细胞模型,探索肌切蛋白对成牙本质细胞MDPC-23增殖、迁移及矿化能力的影响。方法构建沉默表达肌切蛋白的慢病毒载体质粒,转染MDPC-23细胞,获得肌切蛋白沉默表达的稳定转染细胞模型。空载体转染细胞及未转染细胞作为对照组,蛋白印迹法(Western blot)检测肌切蛋白干扰效率,细胞计数(CCK-8)法检测细胞的增殖情况,Transwell小室实验观察细胞迁移率,碱性磷酸酶(ALP)试剂盒及茜素红染色检测细胞矿化差异。采用单因素方差分析对数据进行统计分析,LSD-t检验进行两两比较。结果成功构建肌切蛋白沉默表达的细胞模型,CCK-8结果显示沉默肌切蛋白后,MDPC-23细胞的增殖率在48和72 h时分别下降26.8%(F_(48h)=11.025,P=0.01)和26.5%(F_(72h)=86.205,P<0.001);Transwell结果显示,沉默肌切蛋白后,MDPC-23细胞迁移能力降低61.2%(F=6.005,P=0.008);干扰肌切蛋白后MDPC-23细胞ALP活性明显升高,约为空载体组的4倍(ALP_(干扰组)=568.43 U/gprot,ALP_(空载体组)=142.56 U/gprot,ALP_(空转染组)=118.16 U/gprot;F=49.002,P<0.001),矿化结节形成量高于对照组。结论肌切蛋白可能参与成牙本质细胞MDPC-23的增殖、迁移及矿化过程。

Objective To explore the effect of adseverin on proliferation,migration and mineraliza-tion of MDPC-23 cells. Methods The recombinant lentivirus vector was constructed to knockdown adse-verin and transfected into MDPC-23 cells. Cells transfected with non-targeting shRNA or without shRNA （mock transfection）were used as negative control. Western blot was applied to detect the expression of adseverin. Cell proliferation was examined by CCK-8 method and the migration ability was assayed by Transwell. Alkaline phosphatase activity and Alizarin red staining assay were used to test the effect of adseverin on the mineralization of MDPC-23 cells. The level of significance was determined by One-Way ANOVA followed by LSD-t test for a multiple comparison procedure. Rusults Adseverin expression was significantly deceased compared with the control. Silencing adseverin in MDPC-23 significantly hin-dered cell proliferation by approximately 26.8%（F48 h=11.025,P=0.01）,26.5%（F72 h=86.205,P〈0.001）followed by 48 h and 72 h of culture in growth medium. The number of cells transferred through the Transwell membrane in shRNA-Adseverin group was dramatically reduced by 61.2%（F=6.005,P=0.008）when compared with control. Cells transfected by adseverin shRNA displayed higher level of ALP activity（ALPshRNA-Ads=568.43 U/gprot,ALPshRNA-Ctrl=142.56 U/gprot,ALPMDPC-23=118.16 U/gprot;F=49.002, P〈0.001）and mineralized nodule formation. Conclusion Adseverin might play a crucial role in the pro-liferation,migration and mineralization in MDPC-23 cells.