脾酪氨酸激酶在血小板源性生长因子诱导的肺血管平滑肌细胞表型转换中的作用

目的 观察在血小板源性生长因子(PDGF-BB)诱导增殖的肺血管平滑肌细胞(VSMC)中,脾酪氨酸激酶(syk)的特异性抑制剂piceatannol对VSMC表型转换的影响.方法 以SD大鼠VSMC为基础,设空白对照组(不作任何处理)、对照组(PDGF-BB培养)和药物干预组(PDGF-BB和piceatannol培养).3H]-TdR掺入量测定DNA合成;透射电镜观察细胞超微结构变化;用RT-PCR和Western blot对syk、平滑肌α肌动蛋白(α-SM-actin)及平滑肌22α蛋白(SM22α)表达活性进行检测;激光共聚焦显微镜观察F-actin的改变,并对荧光的改变程度进行定量分析.通过以上方法 观察syk在VSMC增殖和表型转换过程中所起的作用.结果 和空白对照组比较,对照组3H]-TdR掺入量明显升高(2429.25±253.36 vs.242.75±14.33,P<0.01);细胞超微结构呈现增殖状态;syk mRNA(1.70±0.25 vs.1.01±0.12,P<0.05)和蛋白表达水平明显上升;α-SM-actin(0.10±0.00 vs.1.00±0.00,P<0.01)和SM22α(0.18±0.00 vs.1.00±0.01,P<0.01)的mRNA和蛋白表达水平明显下降;细胞骨架蛋白F-actin的荧光强度降低(263.75±19.21 vs.1146.23±62.61,P<0.01).piceatannol可明显抑制这些生物学效应(药物干预组).结论 piceatannol可以抑制血小板源性生长因子介导的VSMC内SM22α和α-SM-aetin的表达,从而抑制VSMC表型的转换,进而抑制VSMC的增殖.说明在VSMC中,PDGF-BB是通过syk信号通路对其表型转换进行调控,进而影响VSMC的迁移和增殖.

Role of spleen tyrosine kinase in phenotypic modulation of vascular smooth muscle cell induced by platelet-derived growth factor-BB

Objective To investigate the role of spleen tyrosine kinase(syk)in the phenotypic modulation induced by platelet-derived growth factor(PDGF-BB)in rat pulmonary vagcular smooth muscle cells(VSMC).Methods Vascular smooth muscles were isolated from pulmonary media of SD rats,cultured,adopted,and divided into 3 groups:blank control group,control group and medicine intervention group.The changes of proliferation and ultrastructure of vascular smooth muscle cells by using3H] thymidine incorporation and electron microscopy.The mRNA and protein expression level of syk,alpha-smooth muscle-actin(α-SM-actin)and smooth muscle protein 22alpha(SM22α)were detected by RT-PCR and Western blotting.The change of fluorescence intensity was detected bv laser scanning confocal microscope.Results Treatment with PDGF-BB for 24 h resulted in a significant increase in 3H] thymidine incorporation(2429.25±253.36 vs.242.75±14.33,P<0.01)and marked change in phenotype and cytoskeleton,the level of average optical density decreased significantly(263.75±19.21 vs.1146.23±62.61.P<0.01).Meanwhile.the mRNA(1.70±0.25 vs.1.01±0.12.P<0.05)and protein leveI of syk significantly increased.the mRNA and protein expression of α-SM-actin(0.10±0.00 vs.1.00±0.00,P<0.01)and SM22α(0.18±0.00 vs.1.00±0.01,P<0.01)significantly decreased in VSMC induced by PDGF-BB.Piceatannol could inhibit significantly these biological effects.Compared with control group,the level of3H]thymidine incorporation(527.00±27.76 vs.2429.25±253.36,P<0.01)was significantly down-regulated and the VSMC presented an apoptotic status in medicine intervention group,the level of average optical density increased significantly(810.65±37.94 vs.263.75±19.21,P<0.01)in medicine intervention group.Meanwhile,the mRNA(0.36±0.07 vs.1.70±0.25,P<0.01)and protein level of syk significantly decreased.The mRNA and protein levels of α-SM-actin(0.22±0.00 vs.0.10±0.00,P<0.01)and SM22α(0.31±0.00 vs.0.18±0.00,P<0.01)were significantly higher in medicine intervention group than in control group.The level of average optical density increased significantly (810.65±37.94 vs.263.75±19.21,P<0.01).Conclusion Syk plays an important role in vascular remodeling by changing the phenotypes and cytoskeleton of VSMC stimulated by PDGF-BB.