小干扰RNA沉默Toll样受体2/4基因对成骨细胞迁移和增殖的影响

Knockdown of Toll-like receptor 2/4 by small interfering RNA suppresses the migration of osteoblasts

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.

Objective 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.