Roscovitine对TNF-α诱导的大鼠血管平滑肌细胞增殖及细胞周期的影响
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摘要
目的观察Roscovitine对TNF-α诱导的大鼠血管平滑肌细胞(VSMC)增殖及细胞周期的影响。方法组织贴块法培养大鼠VSMC细胞,采用TNF-α诱导其增殖,加入不同浓度的Roscovitine预处理15 h,将细胞分为:对照组、TNF-α组、Roscovitine 5、10、15、30μmol·L~(-1)组。MTT比色法检测细胞增殖活性;Western blot检测增殖细胞核抗原(PCNA);流式细胞仪检测细胞周期;荧光定量RT-PCR及Western blot检测细胞周期蛋白(Cyclin A、Cyclin B、Cyclin D、Cyclin E)、细胞周期蛋白依赖激酶(CDK4、CDK5)、细胞周期抑制蛋白(p53、p21、p27)的表达。结果 Roscovitine能抑制VSMC增殖;抑制细胞周期从G_0/G_1期向S期转化。与TNF-α组比较,Roscovitine 5、10、15、30μmol·L~(-1)组能降低细胞周期蛋白Cyclin A、Cyclin B、Cyclin D、Cyclin E蛋白表达,降低细胞周期蛋白依赖激酶CDK4、CDK5蛋白表达,升高细胞周期抑制蛋白p53、p21、p27蛋白表达(P<0.05)。结论 Roscovitine可抑制大鼠VSMC细胞周期进程及增殖活性。
Aim To observe the effect of roscovitine on TNF-α-induced proliferation and cell cycle of rat vascular smooth muscle cells(VSMCs).Methods VSMCs were isolated and cultured using tissue explant method. Tumor necrosis factor-alpha(TNF-α) was used to induce the proliferation of VSMCs. VSMCs were divided into control group, TNF-α group and roscovitine 5, 10, 15, 30 μmol·L~(-1) group. Cell proliferation was detected with MTT, and PCNA expression was tested. Flow cytometry was used to detect the cell cycle. The expressions of cyclin proteins(Cyclin A, Cyclin B, Cyclin D, Cyclin E), cyclin-dependent kinase proteins(CDK4, CDK5), and cell cycle inhibitory proteins(p53, p21, p27) were examined by real-time reverse transcription PCR(qRT-PCR) and Western blot.Results Roscovitine could inhibit the proliferation of VSMCs. Roscovitine inhibited transformation of cell cycle from G_0/G_1 phase to S phase. Compared with TNF-α group, the expressions of Cyclin A, Cyclin B, Cyclin D, Cyclin E protein decreased, the expressions of CDK4 and CDK5 protein decreased, while the expression of p53, p21 and p27 protein in cell cycle increased of roscovitine 5, 10, 15, 30 μmol·L~(-1) group(P<0.05).Conclusion Roscovitine inhibits the cell cycle progression and proliferative activity of rat VSMCs.
Aim To observe the effect of roscovitine on TNF-α-induced proliferation and cell cycle of rat vascular smooth muscle cells(VSMCs).Methods VSMCs were isolated and cultured using tissue explant method. Tumor necrosis factor-alpha(TNF-α) was used to induce the proliferation of VSMCs. VSMCs were divided into control group, TNF-α group and roscovitine 5, 10, 15, 30 μmol·L~(-1) group. Cell proliferation was detected with MTT, and PCNA expression was tested. Flow cytometry was used to detect the cell cycle. The expressions of cyclin proteins(Cyclin A, Cyclin B, Cyclin D, Cyclin E), cyclin-dependent kinase proteins(CDK4, CDK5), and cell cycle inhibitory proteins(p53, p21, p27) were examined by real-time reverse transcription PCR(qRT-PCR) and Western blot.Results Roscovitine could inhibit the proliferation of VSMCs. Roscovitine inhibited transformation of cell cycle from G_0/G_1 phase to S phase. Compared with TNF-α group, the expressions of Cyclin A, Cyclin B, Cyclin D, Cyclin E protein decreased, the expressions of CDK4 and CDK5 protein decreased, while the expression of p53, p21 and p27 protein in cell cycle increased of roscovitine 5, 10, 15, 30 μmol·L~(-1) group(P<0.05).Conclusion Roscovitine inhibits the cell cycle progression and proliferative activity of rat VSMCs.
引文
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[9] 赵京山, 孙佳欢, 于琨, 等. Roscovitine通过影响核因子κB活化抑制大鼠颈动脉内膜损伤导致的炎性增生[J]. 中国病理生理杂志, 2017, 33(2): 233-8.[9] Zhao J S,Sun J H,Yu K, et al. Roscovitine inhibits inflammatory hyperplasia of carotid artery intima in rats via suppressing nuclear factor-KB activation[J]. Chin J Pathophysiol, 2017, 33(2): 233-8.
[10] 杨冬梅, 阳巍, 邱飞, 等. 姜黄素烟酸酯对血管平滑肌细胞增殖及ERK1/2信号通路的影响[J]. 中国药理学通报, 2016 , 32(11): 1526-30.[10] Yang D M, Yang W, Qiu F, et al. Effects of Cur Tn on proliferation of VSMC[J]. Chin Pharmacol Bull, 2016, 32(11): 1526-30.
[11] Liu Y, Li Y F, Chang H, et al. Roscovitine protects from arterial injury by regulating the expressions of c-Jun and p27 and inhibiting vascular smooth muscle cell proliferation[J]. J Cardiovasc Pharmacol,2017, 69(3):161-9.
[12] 李敬美, 丁圆媛, 潘夕春, 等. 细胞周期调节因子基因在H9c2心肌细胞肥大过程中的时相性表达变化[J]. 中国药理学通报, 2017,33(1):63-8.[12] Li J M, Ding Y Y, Pan X C, et al. Temporal expressions of cell cycle regulators mRNA during hypertrophic process of H9c2 rat cardiomyocytes induced by angiotensin Ⅱ[J]. Chin Pharmacol Bull, 2017,33(1): 63-8.
[13] Ingham M,Schwartz G K. Cell-cycle therapeutics come of age[J]. J Clin Oncol,2017, 35(25):2949-59.
[2] Uryga A K, Bennett M R. Ageing induced vascular smooth muscle cell senescence in atherosclerosis[J]. J Physiol,2016, 594(8):2115-24.
[3] Welt F G, Rogers C. Inflammation and restenosis in the stent era[J]. Arterioscler Thromb Vasc Biol, 2002, 22(11):1769-76.
[4] Cicenas J, Kalyan K, Sorokinas A, et al. Roscovitine in cancer and other diseases[J]. Ann Transl Med, 2015, 3(10):135-47.
[5] Xie T, Hu G, Dong B, et al. Roscovitine protects murine Leydig cells from lipopolysaccharide-induced inflammation[J]. Exp Ther Med, 2017, 13(5):2169-76.
[6] Arisan E D, Obakan P, Coker A, et al. Inhibition of ornithine decarboxylase alters the roscovitine-induced mitochondrial-mediated apoptosis in MCF-7 breast cancer cells[J]. Mol Med Rep, 2012, 5(5):1323-9.
[7] Pizarro J G, Folch J, Junyent F, et al. Antiapoptotic effects of roscovitine on camptothecin-induced DNA damage in neuroblastoma cells[J]. Apoptosis, 2011, 16(5):536-50.
[8] Sroka I M, Heiss E H, Havlicek L,et al. A novel roscovitine derivative potently induces G1-phase arrest in platelet-derived growth factor-bb-activated vascular smooth muscle cells[J]. Mol Pharmacol, 2010, 77(2): 255-61.
[9] 赵京山, 孙佳欢, 于琨, 等. Roscovitine通过影响核因子κB活化抑制大鼠颈动脉内膜损伤导致的炎性增生[J]. 中国病理生理杂志, 2017, 33(2): 233-8.[9] Zhao J S,Sun J H,Yu K, et al. Roscovitine inhibits inflammatory hyperplasia of carotid artery intima in rats via suppressing nuclear factor-KB activation[J]. Chin J Pathophysiol, 2017, 33(2): 233-8.
[10] 杨冬梅, 阳巍, 邱飞, 等. 姜黄素烟酸酯对血管平滑肌细胞增殖及ERK1/2信号通路的影响[J]. 中国药理学通报, 2016 , 32(11): 1526-30.[10] Yang D M, Yang W, Qiu F, et al. Effects of Cur Tn on proliferation of VSMC[J]. Chin Pharmacol Bull, 2016, 32(11): 1526-30.
[11] Liu Y, Li Y F, Chang H, et al. Roscovitine protects from arterial injury by regulating the expressions of c-Jun and p27 and inhibiting vascular smooth muscle cell proliferation[J]. J Cardiovasc Pharmacol,2017, 69(3):161-9.
[12] 李敬美, 丁圆媛, 潘夕春, 等. 细胞周期调节因子基因在H9c2心肌细胞肥大过程中的时相性表达变化[J]. 中国药理学通报, 2017,33(1):63-8.[12] Li J M, Ding Y Y, Pan X C, et al. Temporal expressions of cell cycle regulators mRNA during hypertrophic process of H9c2 rat cardiomyocytes induced by angiotensin Ⅱ[J]. Chin Pharmacol Bull, 2017,33(1): 63-8.
[13] Ingham M,Schwartz G K. Cell-cycle therapeutics come of age[J]. J Clin Oncol,2017, 35(25):2949-59.