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中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (03) : 129 -135. doi: 10.3877/cma.j.issn.1674-1366.2017.03.001

所属专题: 文献

基础研究

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
郭冰1, 蒋宏伟1, 凌均棨1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2017-02-09 出版日期:2017-06-01
  • 通信作者: 凌均棨

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

Bing Guo1, Hongwei Jiang1, Junqi Ling1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2017-02-09 Published:2017-06-01
  • Corresponding author: Junqi Ling
  • About author:
    Corresponding author: Ling Junqi, Email:
引用本文:

郭冰, 蒋宏伟, 凌均棨. 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[J/OL]. 中华口腔医学研究杂志(电子版), 2017, 11(03): 129-135.

Bing Guo, Hongwei Jiang, Junqi Ling. Effect of adseverin on proliferation, migration and mineralization of MDPC-23 cells[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(03): 129-135.

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%(F48 h= 11.025,P= 0.01)和26.5%(F72 h= 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 mineralization of MDPC-23 cells.

Methods

The recombinant lentivirus vector was constructed to knockdown adseverin 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 hindered 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 proliferation, migration and mineralization in MDPC-23 cells.

图1 肌切蛋白在MDPC-23中的表达及与F-actin的共定位关系(× 200)
图2 免疫荧光检测shRNA转染效率(× 40)
图3 Western blot检测shRNA转染后肌切蛋白的表达情况
图4 肌切蛋白干扰后对MDPC-23细胞增殖情况的影响
图5 肌切蛋白干扰对MDPC-23细胞迁移能力的影响
图6 肌切蛋白干扰对MDPC-23细胞矿化结节形成的影响(倒置荧光显微镜× 50)
图7 肌切蛋白干扰对MDPC-23细胞ALP活性的影响
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