整合素β1介导牙龈蛋白酶所致的小鼠成骨细胞周期阻滞  

Integrin β1 mediates gingipain-induced cell cycle arrest in osteoblasts

作　　者：徐娜 吴娟[1] 邱绮虹 张福萍[1] 梁敏[1] Xu Na;Wu Juan;Qiu Qihong;Zhang Fuping;Liang Min(Guanghua School of Stomatology,Hospital of Stomatology,Sun Yat.sen University,Guangdong Provincial Key Laboratory of Stomatology,Guangzhou 510055,China)

机构地区：[1]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,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2018年第4期213-220,共8页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(81170970)

摘　　要：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 integrinβ1(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.

关 键 词：整合素Β1 细胞周期 成骨细胞 慢性牙周炎 牙龈蛋白酶 牙龈卟啉单胞菌 

分 类 号：R781.42[医药卫生—口腔医学]