Toll 样受体配体 CpG 寡脱氧核苷酸调节成骨细胞的增殖和成骨分化。
The Toll-like receptor ligand, CpG oligodeoxynucleotides, regulate proliferation and osteogenic differentiation of osteoblast.
机构信息
Department of Periodontics, School and hospital of Stomatology, Jilin University, 1500 Qinghua Road, Changchun, Jilin, 130021, China.
Department of Orthodontics, Tianjin Key Laboratory of Oral and Maxillofacial Function Reconstruction; Tianjin Stomatological Hospital; Hospital of Stomatology, Nankai University, 75 Dagu North Road, Tianjin, 300041, China.
出版信息
J Orthop Surg Res. 2020 Aug 14;15(1):327. doi: 10.1186/s13018-020-01844-x.
BACKGROUND
This study aimed to investigate the regulation of CpG oligodeoxynucleotides (ODNs) on proliferation and osteogenic differentiation of MC3T3 cells.
METHODS
The laser co-focusing and flow cytometry assay were employed to detect cell uptake of CpG ODN 2006. Twelve ODNs were sythesized, and their effects on proliferation and differentiation were detected by MTT and alkaline phosphatase (ALP) activity assay. Flow cytometry assay was used to examine the regulation of CpG ODN on cell cycle. Quantitative real-time PCR (qRT-PCR) and western blot were used to evaluate the regulation of CpG ODN on mRNA and protein expression of osteogenic differentiation genes.
RESULTS
The phosphorothioate CpG ODN 2006 could efficiently enter the MC3T3 cells in 1 h and locate in the cytoplasm. The MTT assay demonstrated CpG ODNs could promote MC3T3 cell proliferation and differentiation in the early stage, and gradually attenuated along with the increase of treating time, except for BW001 and FC001. qRT-PCR assay demonstrated that all the 12 CpG ODNs could promote the relative expression level of osteogenic differentiated genes, SP7 and OCN. In addition, western blot analysis suggested the CpG ODNs of BW001 and FC001 could increase the protein expression of P27 and Runx2 and decrease the protein expression of cyclin D1.
CONCLUSION
The selected CpGODNs may be a potential gene therapy for bone regeneration of periodontitis.
背景
本研究旨在探讨 CpG 寡脱氧核苷酸（ODN）对 MC3T3 细胞增殖和成骨分化的调节作用。
方法
采用激光共聚焦和流式细胞术检测 CpG ODN 2006 的细胞摄取情况。合成 12 种 ODN，通过 MTT 和碱性磷酸酶（ALP）活性检测试剂盒检测其对细胞增殖和分化的影响。流式细胞术检测 CpG ODN 对细胞周期的调节作用。采用实时定量 PCR（qRT-PCR）和 Western blot 检测 CpG ODN 对成骨分化基因 mRNA 和蛋白表达的调控作用。
结果
硫代磷酸化 CpG ODN 2006 可在 1 h 内有效进入 MC3T3 细胞，并定位于细胞质中。MTT 试验表明，CpG ODN 可促进 MC3T3 细胞在早期增殖和分化，随着处理时间的增加逐渐减弱，但 BW001 和 FC001 除外。qRT-PCR 试验表明，这 12 种 CpG ODN 均可促进成骨分化基因 SP7 和 OCN 的相对表达水平。此外，Western blot 分析表明，BW001 和 FC001 的 CpG ODN 可增加 P27 和 Runx2 的蛋白表达水平，降低细胞周期蛋白 D1 的蛋白表达水平。
结论
所选 CpG ODN 可能是治疗牙周炎骨再生的潜在基因治疗方法。
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