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目的探索Rock信号通路在肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)引起兔基底动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用。方法组织块法原代培养兔基底动脉VSMCs,TNF-α刺激VSMCs,Rho激酶(Rho kinase,Rock)抑制剂Y-27632预处理,CCK-8法检测细胞增殖变化,流式细胞仪检测细胞周期,免疫细胞化学观察增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在VSMCs的定位及表达变化,Western blot法检测细胞PCNA表达量的变化。结果细胞培养24 h后,与阴性对照组比较,20 ng/mL TNF-α处理可以显著增加VSMCs的增殖率(P<0.01),PCNA蛋白表达显著增高(P<0.01),S期+G2/M期细胞比例明显增加(P<0.05);加入Y-27632预处理后,与20 ng/mL TNF-α处理组相比,VSMCs的增殖率受到显著抑制(P<0.01),PCNA蛋白表达明显减少(P<0.05),S期+G2/M期细胞比例明显减少(P<0.05)。结论 TNF-α可促进VSMCs增殖和细胞周期进程,Rock抑制剂可阻滞TNF-α对VSMCs的这一作用,Rock信号通路参与TNF-α诱导的VSMCs增殖和细胞周期进程加速。
Objective To explore the role of Rock signaling pathway in the proliferation of rabbit vascular smooth muscle cells (VSMCs) induced by tumor necrosis factor-α (TNF-α) in rabbits. Methods VSMCs of rabbit basilar artery were stimulated by tissue-block method, VSMCs were stimulated by TNF-α and Y-27632 was pretreated with Rho kinase inhibitor. Cell proliferation was detected by CCK-8 assay. Cell cycle was detected by flow cytometry Immunocytochemistry was used to observe the localization and expression of proliferating cell nuclear antigen (PCNA) in VSMCs. The expression of PCNA was detected by Western blot. Results After cultured for 24 h, the proliferation of VSMCs was significantly increased (P <0.01) and the expression of PCNA protein was significantly increased (P <0.01). Compared with the negative control group, 20 ng / mL TNF- (P <0.05). After pretreatment with Y-27632, the proliferation rate of VSMCs was significantly inhibited (P <0.01) and the expression of PCNA protein was significantly higher than that of 20 ng / mL TNF- (P <0.05). The proportion of cells in S phase and G2 / M phase decreased significantly (P <0.05). Conclusion TNF-α can promote VSMCs proliferation and cell cycle progression. Rock inhibitor can block the effect of TNF-α on VSMCs. Rock signaling pathway is involved in the proliferation and cell cycle progression of VSMCs induced by TNF-α.