CCN3调控牙周膜成纤维细胞的增殖和凋亡

doi:10.19439/j.sjos.2017.05.008

摘要/Abstract

摘要： 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pcDNA.3.1-CCN3并转染人PDLFs过表达CCN3,转染CCN3 siRNA,抑制其表达量。转染Fra-1 siRNA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（qRT-PCR）检测CCN3、Fra-1 mRNA表达水平,Western 印迹法检测CCN3、Fra-1和Bcl-2蛋白表达量。MTT和BrdU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pcDNA.3.1-CCN3的实验中, CCN3 mRNA（P<0.05）和蛋白（P<0.05）表达量显著上升。转染CCN3 siRNA 的实验中,CCN3 mRNA（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。

关键词: CCN3, 牙周膜成纤维细胞, Fra-1, 细胞增殖, 细胞凋亡

Abstract: PURPOSE: To investigate the effect of CCN3 on proliferation and apoptosis in periodontal ligament fibroblasts (PDLFs) and related mechanism. METHODS: Recombinant vector pcDNA.3.1-CCN3 was constructed and transfected into human PDLFs to overexpress CCN3. CCN3 siRNA was transfected to inhibit CCN3. Fra-1 siRNA was transfected into the PDLFs with CCN3 inhibition to realize the inhibition of CCN3 and Fra-1 in the meantime. mRNA expressions of CCN3 and Fra-1 were measured by quantitative real-time PCR (qRT-PCR). The protein expressions of CCN3, Fra-1and Bcl-2 were detected by Western blot. Cell growth and viability and proliferation of PDLFs were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assays; Caspase-3 activity was tested by using the available kit. The data were analyzed with SPSS20.0 software package. RESULTS: The results showed that the mRNA (P<0.05) and protein (P<0.05) expressions of CCN3 were significantly up-regulated in the experimental group of pcDNA.3.1-CCN3 transfection. In addition, the mRNA (P<0.05) and protein (P<0.05) expressions of CCN3 were significantly decreased in the experimental group of CCN3 siRNA transfection. Cell growth (P<0.05) and viability, proliferation (P<0.05) and Bcl-2 (P<0.05) protein expression were increased, while caspase-3 activity (P<0.05) decreased in the PDLFs with CCN3 inhibition. However, CCN3 overexpression exhibited reversed effect. CCN3 overexpression or inhibition could remarkably constrain (P<0.05) or promote (P<0.01) the expression of Fra-1, respectively. Moreover, co-inhibition of CCN3 and Fra-1 could promote apoptosis (P<0.01) and inhibit proliferation (P<0.05) in PDLFs. CONCLUSIONS: The results suggest that inhibition of CCN3 could accelerate proliferation and constrain apoptosis via up-regulating expression of Fra-1 in PDLFs.

Key words: CCN3, Periodontal ligament fibroblasts, Fra-1, Cell proliferation, Cell apoptosis

中图分类号:

R781.4

李凤霞, 王俊, 马玉云. CCN3调控牙周膜成纤维细胞的增殖和凋亡[J]. 上海口腔医学, 2017, 26(5): 504-509.

LI Feng-xia, WANG Jun, MA Yu-yun. CCN3 regulates the proliferation and apoptosis in periodontal ligament fibroblasts[J]. Shanghai Journal of Stomatology, 2017, 26(5): 504-509.

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