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目的:观察体外培养大鼠主动脉平滑肌细胞(VSMC)在TGF-β1刺激下骨膜素(periostin)的表达情况及其与平滑肌细胞迁移、增殖等的关系;观察阿托伐他汀对上述过程的影响并初步探讨其抑制periostin表达的分子机制。方法:采用组织块贴壁培养法进行血管平滑肌细胞的原代培养,取3～6代细胞用于实验。采用10 ng/mL TGF-β1刺激VSMC,并用不同浓度阿托伐他汀(0.1,1.0,10.0μmol/L)以及Rho激酶抑制剂Y-27632、甲羟戊酸(MVA)+10.0μmol/L阿托伐他汀干预,24 h后用RT-PCR及Western印迹法检测VSMC中periostin mRNA和蛋白的表达;用Boyden趋化小室测试细胞迁移情况,用MTT法测定细胞增殖情况。结果:大鼠VSMC在TGF-β1刺激下periostin蛋白表达明显增加(4.158±0.515 vs 0.385±0.031),且VSMC迁移数(25±4 vs 8±2)及增殖率(0.85±0.06 vs 0.32±0.03)明显增加,阿托伐他汀呈浓度依赖性抑制大鼠VSMC在TGF-β1诱导下的periostin表达及抑制VSMC的迁移率及增殖率;Rho激酶抑制剂Y-27632可明显降低大鼠VSMC在TGF-β1诱导下的periostin表达(2.082±0.245);加入MVA后,阿托伐他汀对TGF-β1促进大鼠VSMC中periostin表达的抑制作用明显降低(3.838±0.326)。结论:Periostin可促进大鼠VSMC的迁移和增殖,阿托伐他汀可能是通过抑制MVA等类异戊二烯的生成,阻断Rho/Rho激酶信号通路,进而抑制TGF-β1诱导的VSMC中periostin的表达。
OBJECTIVE: To observe the expression of periostin in VSMCs stimulated by TGF-β1 in vitro and its relationship with the migration and proliferation of smooth muscle cells. To observe the effect of atorvastatin on the above process And preliminary study of its molecular mechanism of inhibiting periostin expression. Methods: The primary culture of vascular smooth muscle cells was carried out by adherent culture method. The cells were cultured for 3 to 6 generations. VSMCs were stimulated with 10 ng / mL TGF-β1 and treated with different concentrations of atorvastatin (0.1, 1.0, 10.0 μmol / L) and Rho kinase inhibitors Y-27632, MVA + 10.0 μmol / L Atorvastatin intervention, 24 hrs later, the expression of periostin mRNA and protein in VSMCs was detected by RT-PCR and Western blotting. The cell migration was tested by Boyden chemotaxis chamber and the cell proliferation was measured by MTT assay. Results: Periostin protein expression was significantly increased in VSMCs treated with TGF-β1 (4.158 ± 0.515 vs 0.385 ± 0.031), VSMC migration (25 ± 4 vs 8 ± 2) and proliferation rate (0.85 ± 0.06 vs 0.32 ± 0.03 ) Atorvastatin inhibited periostin expression in VSMCs induced by TGF-β1 in a concentration-dependent manner and inhibited VSMC migration and proliferation rate in a concentration-dependent manner. Rho kinase inhibitor Y-27632 could significantly reduce the expression of VSMCs in TGF (2.082 ± 0.245). After adding MVA, the inhibitory effect of atorvastatin on periostin expression in VSMCs induced by TGF-β1 was significantly reduced (3.838 ± 0.326). Conclusion: Periostin can promote VSMC migration and proliferation in rat VSMCs. Atorvastatin may inhibit the production of isoprenoids such as MVA and block the Rho / Rho kinase signaling pathway, and then inhibit periostin induced by TGF-β1 expression.