The Toll-like receptor ligand, CpG oligodeoxynucleotides, regulate proliferation and osteogenic differentiation of osteoblast

Abstract

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 P27Kip1 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.