Abstract:

Objective To investigate the effects of casein kinase 2 interacting protein-1 (CKIP-1) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs). Methods The hPDLSCs were obtained by primary culture with periodontal ligament tissues that were collected from normal humans. Then, a lentiviral vector containing a CKIP-1-specific siRNA sequence was constructed, and the transcriptional level of CKIP-1 in hPDLSCs was downregulated after vector infection. The P4 cells were divided into four groups: the control group, negative control group (infected with a control vector), CKIP-siRNA group (infected by a CKIP-1 siRNA lentivirus) and CKIP-1 group (infected by a CKIP-1 overexpression virus). All of the cells were cultured under osteogenic induction for 21 days. Then, alizarin red staining and quantitative determination were performed to detect the osteogenic differentiation ability of the hPDLSCs. In addition, qPCR was used to detect the transcriptional level of osteogenesis-related regulatory factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and receptor activator of nuclear factor kappa-B ligand (RANKL), and the osteogenesis-related regulatory factors of the bone morphogenetic protein (BMP) signaling pathway. Results There were no differences in the indexes between the negative control group and the control group (P > 0.05). Compared with the negative control group, the CKIP-siRNA group demonstrated more mineralized nodules (P < 0.05), significantly increased calcium salt deposition (P < 0.05), and increased mRNA levels of osteogenesis-related regulatory factors, such as Runx2 , ALP, OCN, and RANKL, and the osteogenesis-related regulatory factors of BMP signaling pathway (P < 0.05). Conclusion Downregulation of CKIP-1 could promote the osteogenic differentiation of hPDLSCs, which is related to the transcription level of osteogenic-related regulatory factors.

Key words: periodontitis, periodontal bone loss, casein kinase 2 interacting protein-1, human periodontal ligament stem cells, osteogenic differentiation, siRNA interference, Runt-related transcription factor 2, alkaline phosphatase, osteocalcin, receptor activator ofnuclear factor kappa-B ligand, bone morphogenetic protein signaling pathway

摘要：

目的 探讨酪蛋白激酶2相互作用蛋白1（casein kinase 2 interacting protein-1,CKIP-1）对人牙周膜干细胞（human periodontal ligament stem cells,hPDLSCs）成骨分化能力的影响。方法 收集正常人牙周膜组织,通过组织块培养法分离培养hPDLSCs,将P4代细胞分为空白对照组、阴性对照组（感染对照载体）、CKIP-1 siRNA慢病毒组（感染CKIP-1 siRNA慢病毒）以及CKIP-1组（感染CKIP-1过表达慢病毒）。对各组细胞进行成骨诱导培养21 d,茜素红染色观察细胞矿化结节形成情况,并用分光光度计法对矿化结节进行定量分析,同时利用qPCR技术检测各组Runt相关转录因子2（Runt-related transcription factor 2,Runx2）、碱性磷酸酶（alkaline phosphatase,ALP）、骨钙素（osteocalcin,OCN）、核因子κB受体活化因子配体（receptor activator of nuclear factor kappa-B ligand,RANKL）等,以及骨形态发生蛋白（bone morphogenetic protein,BMP）信号通路成骨相关调控因子的转录水平。结果 阴性对照组与空白对照组各指标间无统计学差异（P > 0.05）;与阴性对照组比较,CKIP-1 siRNA慢病毒组出现更多的矿化结节（P < 0.05）,矿化沉积量明显增多（P < 0.05）,Runx2、ALP、OCN、RANKL等,以及BMP信号通路的转录水平不同程度升高（P < 0.05）。结论 CKIP-1下调可促进hPDLSCs成骨分化能力,这与成骨相关调控因子转录水平有关。

关键词: 牙周炎, 牙周骨量丧失, 酪蛋白激酶2相互作用蛋1, 牙周膜干细胞, 成骨分化, siRNA干扰, Runt相关转录因子2, 碱性磷酸酶, 骨钙素, 核因子κB受体活化因子配体;, 骨形态发生蛋白信号通路