联合基因沉默TLR2、4对于LPA诱导的RASMCs表型转化的影响

Effects of joint gene silence of TLR2 and TLR4 on LPA induced phenotypic modulation of RASMCs

目的应用RNA干扰技术单一或者联合沉默Toll样受体(TLR)2、4基因,探讨其在溶血磷脂酸(LPA)诱导的平滑肌细胞(VSMCs)表型转化中的作用。方法根据文献建立分化表型大鼠VSMCs(RASMCs)培养体系,予TLR2、4特异性小干扰RNA(siRNA)转染RASMCs,LPA 1μmol/L处理4h后,荧光定量RT-PCR法、Western blot法检测分化和去分化细胞表型标志基因平滑肌肌动蛋白(SMA-α)和骨桥蛋白(OPN)基因及蛋白水平的表达。结果 TLR2、4-siRNA分别转染细胞后可显著抑制LPA诱导的RASMCs细胞SMA-α基因、蛋白下调及OPN基因、蛋白上调,TLR2、4联合干扰组对于LPA诱导的RASMCs细胞SMA-α基因、蛋白下调及OPN基因、蛋白上调的抑制作用较TLR2、4单独干扰组进一步加强。结论 TLR2、4信号通路参与了LPA诱导的表型转化,联合干预TLR2、4,有可能成为稳定动脉粥样硬化斑块、抗粥样硬化治疗的一个新途径之一。

Objective To apply the RNA interference technology to singly or jointly to silence toll-like receptor（TLR）-2,4genes and to investigate their effects on lysophosphatidic acid（LPA）induced phenotypic modulation of vascular smooth muscle cells（VSMCs）.Methods The culture system of rat aortic smooth muscle cells（RASMCs）in differentiated phenotype rats was established according to the literature.Then TLR-2and TLR 4specially small interference RNA（TLR2-siRNA,TLR4-siRNA）were tranfected into RASMCs.After the treatment by 1μmol/L of LPA for 4h,the gene and protein levels of SMA-αand OPN as the marker genes for VSMC differentiated and dedifferentiated phenotype were detected by real-time quantitative RT-PCR and Western blot respectively.Results TLR2-siRNA and TLR4-siRNA transfection could significantly inhibit the down-regulation of SMA-αgene and protein,and the up-regulation of OPN gene and protein in LPA induced RASMCs,the inhibiting effect of the joint interference of TLR2 and TLR4on the down-regulation of SMA-αgene and protein,and the up-regulation of OPN gene and protein in LPA induced RASMCs was further strengthened than the single interference of TLR2 and TLR 4.Conclusion The TLR2 and TLR4signal pathway participates in the LPA-stimulated phenotypic modulation of RASMCs.The joint intervention on TLR2 and TLR4might become one of new pathway for stabilizing atherosclerotic plaque and anti-atherosclerotic therapy.