纤维蛋白原诱导大鼠主动脉平滑肌细胞增殖及趋化

Fibrinogen Induces the Proliferation and Chemotaxis of Rat Aorta Smooth Muscle Cell

目的观察不同浓度纤维蛋白原对体外培养的血管平滑肌细胞增殖、趋化和随机迁移的影响,探讨其致动脉粥样硬化的可能作用及其机制。方法采用胶原酶消化法培养大鼠主动脉平滑肌细胞,用BrdU掺入法和细胞计数法观察其增殖能力,刮伤实验及慢速显微摄像技术检测其随机迁移能力,微孔滤膜法观察其趋化性,Western印迹法检测其明胶酶A的表达,明胶酶谱分析其分泌的明胶酶活性。结果细胞计数(r=0.860,P<0.05)和BrdU掺入实验(r=0.880,P<0.05)发现0.2gL～3.2gL纤维蛋白原呈剂量依赖性地促进血管平滑肌细胞增殖,其中0.4、0.8、1.6和3.2gL纤维蛋白原作用48h,细胞计数依次为(6.32±0.39)×107L、(10.63±0.25)×107L、(12.31±0.44)×107L和(13.59±0.43)×107L,BrdU掺入率依次为5.2%±2.2%、17.5%±2.0%、21.5%±2.3%和25.5%±2.5%,与对照组[分别为(5.63±0.34)×107L和2.0%±0.8%]比较差异均有显著性(P<0.01)。微孔滤膜法趋化实验发现0.2gL～3.2gL纤维蛋白原呈剂量依赖性地促进血管平滑肌细胞趋化(r=0.957,P<0.01)。刮伤实验中细胞迁移速度及慢速显微摄像单细胞随机迁移速度在纤维蛋白原组及对照组细胞均无明显差别(P>0.05)。Western印迹及明胶酶谱发现纤维蛋白原促使血管平滑肌细胞明胶酶A的表达及活性上调。结论纤维蛋白原可能通过促进血管平滑肌细胞增殖和趋化在动脉粥样硬化发生过程中起重要作用。

Aim To investigate the effect of fibrinogen on prolfferafion,chemotaxis and migration of vascular smooth muscle cell （VSMC） and to explore the possible effect and mechanism of fibrinogen in the formation of atherosclerosis. Methods Rat aorta smooth muscle cells were primarily cultured. VSMC proliferation was evaluated by BrdU incorporation assay and visual cell counting. VSMC migration and chemotaxis were measured with serape assay, time-lapse micreeinematography and Boyden＇s chemotaxis assay. Glufinase A expression and activity were assesed by Western blotting and zymography techniques. Results BrdU incorporation assay（r = 0.880, P 〈0.05）and visual cell counting （r=0.860, P 〈0.05）showed that fibrinogen stimulated the proliferation of VSMC dose-dependently in the range between 0.2 g/L and 3.2 g/L. The cell counts of VSMC after 48 h incubation with the concentrations of fibrinogen as 0.4, 0.8,1.6 and 3.2 g/L were significantly higher than control group [（6.32±0.39）× 10^7 cells/L, （10.63 ± 0.25）× 10^7 cells/L, （12.31 ± 0.44）× 10^7 cells/L and （13.59 ± 0.43） × 10^7 cells/L vs.（5.63±0.34）×10^7 cells/L,（P〈0.01）]. The BrdU incorporation rates of fibrinogen of 0.4,0.8, 1.6 and 3.2g/Lwere also significantly higher than control group （5.2% ±2.2%,17.5% ± 2.0%,21.5% ±2.3% and 25.5% ± 2.5% vs.2.0% ± 0.8%,P〈0.01）. Fibrinogen stimulated the chemotaxis of VSMC dose-dependently in the range between 0.2 g/L and 3.2 g/L in Boyden＇s chemotaxis assay （r = 0.957, P 〈 0.01）. Fibrinogen had no obvious effeet on VSMC migration in serape assay and on VSMC migration at random direction in Time-lapse micreeinematography （ P 〉0. 5）. Fibrinogen induced the up-regulation of expression and activity of glutinase A. Conclusion By recruiting vascular smooth muscle cells from the media into the intima and promoting them proliferate, fibrinogen may be involved in the pathogenesis of atherosclerosis.