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目的:探讨西洛他唑对原代培养大鼠血管平滑肌细胞迁移能力的影响及其可能的调控作用。方法:实验于2003-03/12在解放军沈阳军区总医院全军心血管病研究所完成。使用胶原酶消化法处理健康雄性SD大鼠,获得原代培养的鼠血管平滑肌细胞,加入终浓度为0.5μmol/L西洛他唑培养72h为西洛他唑组,对照组为加入等量的Dulbecco改良的Eagle培养液培养的血管平滑肌细胞。应用细胞刮伤实验和基质金属蛋白酶活性测定分析西洛他唑对原代培养血管平滑肌细胞迁移能力的影响。应用蛋白质印记杂交方法分析给药前后血管平滑肌细胞内基质金属蛋白酶2,9和细胞外信号调节激酶蛋白的表达情况。结果:①细胞刮伤实验显示,与对照组比较,西洛他唑组血管平滑肌细胞迁移能力明显受抑,迁移距离明显缩短。明胶酶活性分析发现,西洛他唑组细胞基质金属蛋白酶活性明显低于对照组。②蛋白质印迹杂交分析发现,西洛他唑可引起原代培养血管平滑肌细胞迁移能力下降。西洛他唑组细胞内基质金属蛋白酶2,9蛋白表达明显低于对照组(14.34±0.70,12.65±0.98;93.67±1.90,83.67±1.05,t=67.86,85.65,P<0.05)。③蛋白质印迹杂交分析检测显示,西洛他唑组与对照组血管平滑肌细胞内信号调节激酶1,2蛋白表达无明显差异(P>0.05);细胞内磷酸化信号调节激酶蛋白表?
Objective: To investigate the effect of cilostazol on migration of primary cultured rat vascular smooth muscle cells and its possible regulation. METHODS: The experiment was performed at the Institute of Cardiovascular Diseases, PLA General Hospital of PLA Shenyang Military Area Command from March to December 2003. The primary cultured rat vascular smooth muscle cells were treated with collagenase digestion method, and the cilostazol at a final concentration of 0.5μmol / L was cultured for 72h to induce the cilostazol group. The control group was treated with the same amount of Dulbecco’s modified Eagle’s medium cultured vascular smooth muscle cells. The effects of cilostazol on the migration of primary cultured vascular smooth muscle cells were analyzed by cell scratch assay and matrix metalloproteinase activity assay. The protein expression of MMP-2, 9 and extracellular signal-regulated protein kinase in vascular smooth muscle cells before and after administration were analyzed by Western blotting. Results: (1) The cell scratch test showed that compared with the control group, the migration ability of vascular smooth muscle cells in cilostazol group was significantly inhibited and the migration distance was significantly shortened. Gelatinase activity analysis found that the cilostazol group of matrix metalloproteinase activity was significantly lower than the control group. Western blot hybridization analysis found that cilostazol can cause primary cultured vascular smooth muscle cells decreased migration ability. The expression of matrix metalloproteinase 2,9 in cilostazol group was significantly lower than that in control group (14.34 ± 0.70, 12.65 ± 0.98; 93.67 ± 1.90, 83.67 ± 1.05, t = 67.86, 85.65, P <0.05). ③ Western blot analysis showed that there was no significant difference in the expression of signal regulated kinase 1 and 2 between the cilostazol group and the control group (P> 0.05); the phosphorylation signal regulated kinase protein