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目的:探讨CCN3对牙周膜成纤维细胞(periodontal ligament fibroblasts,PDLFs)增殖和凋亡的作用及可能的机制。方法:构建重组载体pc DNA.3.1-CCN3并转染人PDLFs过表达CCN3,转染CCN3 si RNA,抑制其表达量。转染Fra-1 si RNA到抑制CCN3的PDLFs,同时抑制CCN3和Fra-1的表达量。应用实时荧光定量PCR(q RT-PCR)检测CCN3、Fra-1 m RNA表达水平,Western印迹法检测CCN3、Fra-1和Bcl-2蛋白表达量。MTT和Brd U实验分别检测PDLFs的生长活力和增殖能力。试剂盒方法检测caspase-3的活性。采用SPSS20.0软件包对数据进行统计学分析。结果:转染pc DNA.3.1-CCN3的实验中,CCN3 m RNA(P<0.05)和蛋白(P<0.05)表达量显著上升。转染CCN3 si RNA的实验中,CCN3 m RNA(P<0.05)和蛋白(P<0.05)表达量显著下降。CCN3表达量被抑制后,PDLFs的增殖能力(P<0.05)和生长活力(P<0.05)显著上升,caspase-3活性(P<0.05)和Bcl-2蛋白表达量(P<0.05)分别降低和升高。然而,CCN3过表达时作用相反。CCN3过表达或抑制可分别显著降低(P<0.05)或促进(P<0.01)Fra-1的表达量。另外,同时抑制CCN3和Fra-1,可促进PDLFs的凋亡(P<0.01)且抑制其增殖能力(P<0.05)。结论:抑制CCN3可通过上调Fra-1的表达,促进PDLFs的增殖能力并抑制其凋亡。
Objective: To investigate the effect and possible mechanism of CCN3 on proliferation and apoptosis of periodontal ligament fibroblasts (PDLFs). Methods: Recombinant vector pcDNA3.1-CCN3 was constructed and transfected into human PDLFs overexpressing CCN3, transfected with CCN3 si RNA to inhibit its expression. Transfection of Fra-1 si RNA to CCL3-inhibited PDLFs inhibited both CCN3 and Fra-1 expression. The expression of CCN3 and Fra-1 m RNA were detected by real-time fluorescence quantitative PCR (q RT-PCR). The expression of CCN3, Fra-1 and Bcl-2 protein were detected by Western blotting. MTT and BrdU assays were used to detect the viability and proliferation of PDLFs. Kit assay caspase-3 activity. SPSS20.0 software package for statistical analysis of the data. Results: The expression of CCN3 m RNA (P <0.05) and protein (P <0.05) were significantly increased in pcDNA3.1-CCN3 transfected cells. In CCN3 siRNA transfection, the expression of CCN3 m RNA (P <0.05) and protein (P <0.05) decreased significantly. The proliferation of PDLFs (P <0.05) and the growth vigor (P <0.05) were significantly increased when the CCN3 expression was inhibited. The activity of caspase-3 (P <0.05) and the expression of Bcl- And rise. However, the effect of CCN3 overexpression is reversed. The overexpression or inhibition of CCN3 could significantly reduce (P <0.05) or promote (P <0.01) the expression of Fra-1. In addition, simultaneous inhibition of CCN3 and Fra-1 promoted PDLFs apoptosis (P <0.01) and inhibited their proliferation (P <0.05). Conclusion: Inhibition of CCN3 can promote the proliferation and inhibit the apoptosis of PDLFs by up-regulating the expression of Fra-1.