摘要目的 观察利用RNA干扰技术特异性阻断Toll样受体2/4(TLR2/TLR4)对大鼠成骨细胞迁移及增殖的影响.方法 构建TLR2/TLR4的小干扰RNA (siRNA),转染成骨细胞后采用反转录-聚合酶链反应(RT-PCR)及Western blot法检测TLR2/TLR4的mRNA及蛋白的含量.采用Transwell小室法及噻唑蓝(MTF)法检测转染组和非转染组成骨细胞的迁移能力以及增殖能力,同时采用Western blot法检测各组细胞内核因子-κB (NF-κB)的含量.结果 针对TLR2及TLR4的siRNA可有效抑制其mRNA的表达,抑制率分别达到了94％及84％,同时也抑制了受体蛋白的表达,使其降低至对照组的48％及65％.当TLR2及TLR4表达受到抑制后成骨细胞的迁移能力下降至对照组的76％及59％,但细胞增殖比较差异无统计学意义(P＞0.05),同时细胞内NF-κB活性降低,细胞核内NF-κB p65的含量降至对照组的47％及75％.结论 siRNA能够有效沉默TLR2/TLR4基因的表达,减弱细胞内NF-κB的活化,抑制成骨细胞迁移,表明成骨细胞的迁移通过TLR2/TLR4及NF-κB途径.

abstractsObjective To explore the effects of Toll-like receptor 2/4 (TLR2/TLR4) on the migration and proliferation of osteoblasts.Methods The expression of TLR2/TLR4 in osteoblasts was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.Then the expression of TLR2/TLR4 in osteoblasts was suppressed using RNA interference technique.The influences on the migration and proliferation were analyzed by Transwell chamber assay and methyl thiazol tetrazolium (MTT) assay.Also,the effects of TLR2/TLR4 on the expression of nuclear factor-κB (NF-κB) p65 were detected.Results As shown by RT-PCR and Western blotting,the expression of TLR2/TLR4 significantly decreased in the TLR2/TLR4 small interfering RNA (siRNA)-transfected group.The inhibition rate of mRNA reached 94％ and 84％ respectively.The protein level of TLR2 and TLR4 was reduced to 48％ and 65％ respectively.The number of migrating cells in the TLR2-siRNA and TLR4-siRNA transfected groups was decreased to 76％ and 59％ compared to the control group.But the proliferation of osteoblasts was similar between those groups.Meanwhile,the siRNA-transfected group had lower expression level of NF-κB p65 in nucleus than the control group.The amount of NF-κB p65 in the nucleus was reduced to 47％ and 75％ in the TLR2-siRNA and TLR4-siRNA transfected groups compared to the control group.Conclusion siRNA inhibits the expression of TLR2/TLR4 effectively,weakens the activation of the NF-κB,and then inhibits the migration of the osteoblast.This study implicated that TLR2/TLR4 and NF-κB pathway may be involved in the migration of osteoblasts.