Objective: To investigate the role of NOV/CCN3 in regulating the proliferation of mesenchymal stem cells (MSCs) and its regulatory mechanism and assess the value of CCN3 as a proliferative factor in bone tissue engineering.
Methods: Mouse embryonic fibroblasts (MEFs) were used as the MSC model, in which CCN3 expression was up-regulated and downregulated by transfection with the recombinant adenovirus vectors Ad-CCN3 and Ad-siCCN3, respectively. Flow cytometry was used to analyze the changes in cell cycle and apoptosis of the transfected cells. Western blotting was used to detect the expression levels of the proliferation indicators (PCNA, cyclin E, and cyclin B1) and the apoptosis indicators (Bax and Bcl-2) to assess the effect of modulation of CCN3 expression on MEF proliferation and apoptosis. CCN3 protein secretion by the cells was detected using ELISA. RT-qPCR and Western blotting were employed to analyze the changes in the expressions of Notch1, ligand DLL1, the downstream key proteins or genes (Hey1, P300, H3K9) and MAPK pathway-related proteins ERK1+2 and p-ERK1+2.
Results: Flow cytometry showed that compared with the control cells, MEFs transfected with Ad-CCN3 exhibited significantly increased cell proliferation index (P < 0.01) and lowered cell apoptosis rate (P < 0.05) with obviously enhanced expressions of PCNA, cyclin E and Bcl-2 proteins (P < 0.05). The results of RT-qPCR and Western blotting demonstrated that CCN3 overexpression significantly promoted the expression of Notch1 in the Notch signaling pathway (P < 0.001), inhibited the expressions of DLL1, Hey1, P300, and H3K9 (P < 0.05), and increased the protein expressions of ERK1+2 and P-ERk1+2 in the MAPK pathway (P < 0.01).
Conclusions: CCN3 over-expression promotes the proliferation and inhibits apoptosis of MEFs possibly by inhibiting the classical Notch signaling pathway and activating the MAPK pathway via binding to Notch1, suggesting the potential value of CCN3 as a proliferative factor of MSCs in bone tissue engineering.
目的: 探究肾母细胞瘤过表达因子（NOV/CCN3）对间充质干细胞增殖的影响及调控机制，为CCN3能否作为促增殖因子在骨组织工程的应用提供理论依据。
方法: 以小鼠成纤维细胞（MEFs）为间充质干细胞模型，利用重组腺病毒Ad-CCN3和AdsiCCN3分别上调、下调MEFs中CCN3表达；流式细胞术检测不同处理组细胞周期、凋亡变化情况；Western blot检测增殖指标PCNA、周期蛋白cyclin E、cyclin B1以及凋亡指标Bax、Bcl-2蛋白表达量，明确CCN3对MEFs增殖和凋亡的影响；ELISA检测CCN3蛋白分泌水平；RT-qPCR和Western blot检测Notch信号通路经典受体Notch1、配体DLL1、下游关键蛋白和/或基因Hey1、p300、H3K9，以及MAPK通路相关蛋白ERK1+2和p-ERK1+2表达变化情况。
结果: 流式细胞术结果显示：与对照组相比，Ad-CCN3组细胞增殖指数增加（P < 0.01），细胞凋亡百分比减少（P < 0.05）；且相关增殖凋亡指标PCNA、cyclin E、Bcl-2蛋白表达增加（P < 0.05）；RT-qPCR和Western blot结果显示过表达CCN3明显促进Notch信号通路中Notch1表达（P < 0.01），但抑制DLL1和Hey1、p300、H3K9等表达（P < 0.05）；而MAPK通路中ERK1+2和p-ERK1+2蛋白表达增加（P < 0.01）。
结论: CCN3可能通过与Notch1结合，抑制经典Notch信号通路，激活MAPK通路，促进MEFs增殖、抑制细胞凋亡，从而增加MEFs细胞数量，提示CCN3具有作为间充质干细胞的促增殖因子在骨组织工程发挥作用的潜在价值。
Keywords: CCN3; Notch; cell apoptosis; cell proliferation; embryonic fibroblasts; mesenchymal stem cells.