雷帕霉素脂质体对ox-LDL诱导的血管平滑肌细胞迁移的抑制作用

Inhibitory effect of rapamycin liposomes on ox-LDL induced vascular smooth muscle cell migration

[目的]探讨雷帕霉素脂质体(RL)对氧化型低密度脂蛋白(ox-LDL)诱导的人主动脉血管平滑肌细胞(HA-VSMC)迁移的影响及其与S100钙结合白蛋白A4(S100A4)相关的作用机制。[方法]使用敲低S100A4基因的慢病毒转染HA-VSMC,随后加入嘌呤霉素筛选S100A4基因敲低的稳定株。50 mg/L ox-LDL处理HA-VSMC,加入不同剂量的RL(3、6及12 mg/L),观察处理前后对细胞迁移的影响。采用细胞划痕法、Transwell实验检测细胞迁移,Western blot检测S100A4、磷酸化磷脂酰肌醇3激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、Ⅰ型胶原蛋白(COLⅠ)、波形蛋白的表达。[结果]ox-LDL处理细胞48 h后,与空白对照组相比,S100A4、p-PI3K、p-Akt、p-mTOR、COLⅠ及波形蛋白的表达明显升高(P<0.05),细胞迁移速度明显加快(P<0.05)。与ox-LDL组相比,不同剂量的RL处理48 h后显著抑制S100A4、p-PI3K、p-Akt、p-mTOR、COLⅠ及波形蛋白的表达并显著抑制细胞迁移(P<0.05),其中6 mg/L、12 mg/L RL的抑制作用更明显(P<0.05)。敲低S100A4基因后细胞迁移率显著降低(P<0.05)。[结论]RL能显著抑制ox-LDL诱导的HA-VSMC迁移,可能与RL下调S100A4、p-PI3K、p-Akt、p-mTOR、COLⅠ及波形蛋白的表达相关。

Aim To explore the effects of rapamycin liposomes(RL)on the migration of human aortic vascular smooth muscle cells(HA-VSMC)induced by oxidized low density lipoprotein(ox-LDL)and its mechanism related to S100 calcium binding protein A4(S100A4).Methods Vascular smooth muscle cells were transfected with lentivirus knockdown S100A4 gene,and then added puromycin to screen stable strain of S100A4 gene knockdown.Vascular smooth muscle cells were treated with 50 mg/L ox-LDL,and different doses of RL(3,6 and 12 mg/L)were added to observe the effect on cell migration before and after treatment.Cell migration was detected by cell scratch method and Transwell,and the expression of S100A4,phosphorylated phosphatidylinositol 3-kinase(p-PI3K),phosphorylated protein kinase B(p-Akt),phosphorylated mammalian target of rapamycin(p-mTOR),typeⅠcollagen protein(COLⅠ),and vimentin were detected by Western blot.Results After 48 h treatment with ox-LDL,compared with the blank control group,the expression of S100A4,p-PI3K,p-Akt,p-mTOR,COLⅠand vimentin was significantly increased(P<0.05),and the speed of cell migration was significantly accelerated(P<0.05).Compared with ox-LDL group,different doses of RL significantly inhibited the expression of S100A4,p-PI3K,p-Akt,p-mTOR,COLⅠand vimentin and significantly inhibited cell migration after 48 h treatment(P<0.05),of which 6 mg/L and 12 mg/L of RL had more significant inhibitory effects(P<0.05).After S100A4 gene knockdown,the cell migration rate was significantly reduced(P<0.05).Conclusion RL can significantly inhibit the migration of HA-VSMC induced by ox-LDL,which may be related to the down-regulation of S100A4,p-PI3K,p-Akt,p-mTOR,COLⅠand vimentin expression by RL.