The Influence Of MT01 On The Expression Level Of Toll-like Receptor 7 And 9 In Human Osteoblast-like Cell Line MG63

Toll-like receptors(Toll like receptors, TLRs) is a kind of pattern recognition receptors, firstly expressed on immune cells. Recent studies have found that there is also the expression of TLRs within the osteogenic cells. And studies have shown that, TLRs can regulate bone remodeling process into osteogenic differentiation of bone cells. Wherein the activation of TLR7,9 can increase the expression of interleukin-1(Interleukin, IL-1), tumor necrosis factor(tumor necrosis factorα, TNF-α),thus activating osteoclasts, promoting bone resorption and inhibit bone formation.MT01 is a single-stranded DNA which is designed and synthesized based on human mitochondrial DNA sequences, belongs to an immune-suppressive ODN(oligodeoxynucleotides, ODN). Our previous experiments have confirmed, MT01 can promote osteogenic cells differentiation into osteoblasts, inhibit rat alveolar bone absorption leaded by periodontitis. By reducing the release of inflammatory cytokines in the immune cells, inhibit of the activation of immune response mediated by TLR7 and 9. However, whether the expression of TLR7,9 itself is suppressed is not clear in the process. Our study is to detect the expression of TLRs in human osteoblast-like cell line MG63 on the basis of previous studies. And to further investigate the effect ofMT01 in TLR7 and 9 expression of a gene and the protein level, laying the foundation for future in-depth study the effect and mechanism of MT01 inhibiting alveolar bone resorption, promoting bone formation.Objective: To detect the expression of TLRs in human osteoblast-like cell line MG63. By checking the expression of TLR7 and9 in the levels of m RNA and protein after MT01 roling in human osteoblast-like cell line MG63, preliminary studying the influence of MT01 on the expression level of TLR7 and 9 m RNA and protein.Laying the foundation for further elaboration the immunological mechanisms of MT01 regulate bone remodeling.Methods: First, using the RT-PCR method to screen the TLR1-10 m RNA expression of human osteoblast-like cell line MG63. Using Real time PCR method to detect the expression of TLR7 and 9 m RNA at12 hours, 24 hours and 48 hours after MT01 effecting on MG63 cells;Immunofluorescence staining and Western Blot assay detecting TLR7 and9 protein expression within the cells. Test results using SPSS 17.0statistical software, application ANOVA statistical analysis, P <0.05 was considered statistically significant.Results: MG63 cells express TLR1-10 m RNA, wherein the TLR2,5and 10 m RNA expression are low, the TLR1,3,4,6,7,8 and 9 m RNA expression are high. The expression of TLR7,9 m RNA and protein are significantly inhibited after MT01 acting on and this inhibition is noapparent time-dependent. In 12 hours and 24 hours groups, the TLR7 m RNA and protein expression are significantly reduced, there is a statistically significant difference compared with control group; the expression of TLR7 m RNA and protein in the 48 hours group increased slightly compared with the previous two, but still below the control group,with statistical difference compared with the control group. In 12 hours,24hours, 48 hours groups,the expression of TLR9 m RNA and protein are lower than the control group, and significant difference compared with the control group.Conclusion:(1) MG63 cells express TLR1-10 m RNA, wherein the TLR2,5 and 10 m RNA expression are low, the TLR1,3,4,6,7,8 and 9m RNA expression are high.(2) The expression of TLR7,9 m RNA and protein are significantly inhibited after MT01 acting on.