目的：探讨Toll样受体 (Toll like receptors, TLR)对人牙周膜干细胞成骨分化的影响及其可能的分子机制。方法:应用实时荧光定量聚合酶链式反应（real-time polymerase chain reaction, real-time PCR)及流式细胞术检测TLR在人牙周膜干细胞 (human periodontal ligament stem cells, hPDLSCs)上的表达。在成骨培养基中加入多种浓度的不同TLR配体培养7~14 d，检测TLR对hPDLSCs成骨的影响。通过Western blotting方法检测TLR配体刺激hPDLSCs 7 d后，丝裂原活化蛋白激酶 (mitogen activated protein kinase, MAPK)及蛋白激酶B (protein kinase B, PKB or AKT) 磷酸化水平的变化及runt相关转录因子2 (runt related transcription factor 2, Runx2)的变化，并对比加入MAPK及AKT抑制剂后Runx2的变化, 探究TLR是否通过影响下游的MAPK及AKT的活化而影响hPDLSCs的成骨分化。结果:TLR1、TLR3、TLR4、TLR6在hPDLSCs上表达较高, 不同TLR的阳性细胞百分比为TLR1 2.82%±0.68%，TLR2 1.26%±0.09%，TLR3 13.23%±2.05%，TLR4 3.64%±0.79%，TLR6 3.21%±1.64%，其趋势与TLR 在hPDLSCs中mRNA表达相一致。高浓度的TLR配体 (PolyI:C 10 mg/L, LPS 10 mg/L , Pam3CSK4 1 mg/L, FSL-1 50 μg/L) 刺激hPDLSCs可减少矿化结节的形成, 减弱ALP染色及降低ALP活性。高浓度TLR配体刺激还可下调细胞外调节蛋白激酶（extracellular regulated protein kinases, ERK)、P38、c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK)及AKT的磷酸化水平, 伴随Runx2的表达水平下调, 应用MAPK及AKT抑制剂也可下调Runx2的表达。结论:高浓度的TLR配体刺激可抑制hPDLSCs的成骨分化, 这种抑制作用和MAPK及AKT磷酸化降低相关。

Objective： To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism. Methods: Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium. Results: Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125，SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression. Conclusion: Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.