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中华口腔医学研究杂志(电子版) ›› 2018, Vol. 12 ›› Issue (04) : 213 -220. doi: 10.3877/cma.j.issn.1674-1366.2018.04.003

所属专题: 文献

基础研究

整合素β1介导牙龈蛋白酶所致的小鼠成骨细胞周期阻滞
徐娜1, 吴娟1, 邱绮虹1, 张福萍1, 梁敏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:2018-03-08 出版日期:2018-08-01
  • 通信作者: 梁敏
  • 基金资助:
    国家自然科学基金(81170970)

Integrin β1 mediates gingipain - induced cell cycle arrest in osteoblasts

Na Xu1, Juan Wu1, Qihong Qiu1, Fuping Zhang1, Min Liang1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat- sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2018-03-08 Published:2018-08-01
  • Corresponding author: Min Liang
  • About author:
    Corresponding author: Liang Min, Email:
引用本文:

徐娜, 吴娟, 邱绮虹, 张福萍, 梁敏. 整合素β1介导牙龈蛋白酶所致的小鼠成骨细胞周期阻滞[J]. 中华口腔医学研究杂志(电子版), 2018, 12(04): 213-220.

Na Xu, Juan Wu, Qihong Qiu, Fuping Zhang, Min Liang. Integrin β1 mediates gingipain - induced cell cycle arrest in osteoblasts[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(04): 213-220.

目的

探讨整合素β1(Itgb1)在介导牙龈蛋白酶所致的小鼠成骨细胞周期阻滞中的作用。

方法

8.348 U/L牙龈蛋白酶处理MC3T3-E1细胞12、24、36、48、60、72 h,细胞计数试剂盒(CCK-8)检测细胞增殖活性,含核糖核酸的碘化丙啶(PI/Rnase)染色检测细胞周期G0/G1、S、G2/M各期分布。蛋白免疫印迹法(Western blot)检测cyclin D1和CDK4蛋白表达水平。携带Itgb1基因的慢病毒转染MC3T3-E1细胞,构建Itgb1过表达稳转细胞株。所有数据采用SPSS 22.0统计软件包进行统计学分析。

结果

CCK-8结果显示,8.348 U/L牙龈蛋白酶处理成骨细胞12 ~ 72 h持续抑制细胞增殖。流式检测结果发现,牙龈蛋白酶作用细胞12 h,停滞在G0/G1期的细胞比例由对照组(62.0 ± 2.0)%升至(88.2 ± 2.3)%,差异有统计学意义(F= 35.218,P<0.001),S期细胞比例由对照组(36.8 ± 6.2)%降至(5.9 ± 2.3)%,差异有统计学意义(F= 33.980,P<0.001)。Western blot结果表明,cyclin D1和CDK4的蛋白表达水平与对照组相比分别下调了65.0%(Fcyclin D1=60.294,Pcyclin D1<0.001)和40.3%(FCDK4=19.212,PCDK4= 0.002)。同时牙龈蛋白酶降低Itgb1表达,过表达Itgb1部分逆转牙龈蛋白酶所致的细胞周期阻滞,S期细胞比例由牙龈蛋白酶处理空载体组的(5.8 ± 1.1)%恢复至(14.4 ± 3.1)%,差异有统计学意义(F= 39.226,P= 0.017),cyclin D1和CDK4蛋白下调水平亦得到部分逆转(Fcyclin D1= 61.740,Pcyclin D1= 0.033;FCDK4= 41.635,PCDK4= 0.014)。

结论

Itgb1介导牙龈蛋白酶所致的小鼠成骨细胞细胞周期阻滞。

Objective

To investigate the effect of integrin β1 (Itgb1) in mediating gingipain-induced cell cycle arrest in osteoblasts.

Methods

MC3T3-E1 cells were treated with 8.348 U/L gingipains from 12 to 72 h. Cell counting kit-8 (CCK-8) assay and Propidium Iodide/Rnase (PI/Rnase) staining were used to detect the proliferation activity and cell cycle distribution of G0/G1, S, G2/M phases. The expression of cyclin D1 and CDK4 were evaluated by western blotting after gingipain treatment for 12 h. MC3T3-E1 cells were transducted with lentivirus encoding mouse Itgb1 to overexpress Itgb1. All data were analyzed by SPSS 22.0 software package.

Results

CCK-8 assay showed that 8.348 U/L gingipain treatment inhibited cellular proliferation from 12 to 72 h. The percentage of cells in G0/G1 phase was increased from (62.0 ± 2.0) % to (88.2 ± 2.3) % (F = 35.218, P<0.001) , and the percentage of cells in S phase was decreased from (36.8 ± 6.2) % to (5.9 ± 2.3) % (F= 33.980, P<0.001) compared to control cells. Western blotting showed the protein expression of cyclin D1 and CDK4 were reduced by about 65.0% (Fcyclin D1= 60.294, Pcyclin D1<0.001) and 40.3% (FCDK4= 19.212, PCDK4= 0.002) compared to control cells. Meanwhile, gingipains reduced Itgb1 protein level, overexpression of Itgb1 partially reversed gingipain-induced cell cycle arrest. The percentage of cells in S phase was significantly higher in LV-Itgb1 cells (14.4 ± 3.1) % than that in LV-Ctrl cells (5.8 ± 1.1) % upon gingipain treatment (F= 39.226, P= 0.017) . Similar effects of overexpression of Itgb1 were observed in cyclin D1 and CDK4 down-regulation (Fcyclin D1= 61.740, Pcyclin D1= 0.033; FCDK4= 41.635, PCDK4= 0.014) .

Conclusion

Itgb1 is involved in mediating cell cycle arrest induced by gingipains in osteoblasts.

图1 牙龈蛋白酶抑制MC3T3-E1细胞增殖
图2 PI/Rnase染色流式细胞仪检测牙龈蛋白酶对MC3T3-E1细胞细胞周期影响
图3 牙龈蛋白酶下调MC3T3-E1细胞cyclin D1和CDK4蛋白表达水平
图4 赖氨酸特异性蛋白酶(Kgp)阻滞MC3T3-E1细胞进入S期作用显著
图5 赖氨酸特异性蛋白酶(Kgp)显著下调MC3T3-E1细胞cyclin D1和CDK4蛋白表达水平
图6 牙龈蛋白酶降低MC3T3-E1细胞整合素β1(Itgb1)表达
图7 过表达整合素β1(Itgb1)部分逆转牙龈蛋白酶所致的细胞cyclin D1和CDK4表达下调
图8 过表达整合素β1(Itgb1)部分逆转牙龈蛋白酶所致的成骨细胞细胞周期阻滞
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