血管平滑肌细胞细胞周期素依赖性激酶抑制因子的调节

The regulated pathways of cyclin-dependent kinases inhibitors in vascular smooth muscle cell proliferation

目的 阐明有丝分裂源激活的蛋白激酶 (MAPK)和磷脂酶C(PLC)通路在调节细胞周期素依赖性激酶抑制因子 (CKI)p2 7,p2 1,p5 7蛋白表达量中的作用及其对血管平滑肌细胞 (VSMC)增殖的影响。方法 分离SD大鼠主动脉中层平滑肌 ,贴壁法培养平滑肌细胞 ,无血清培养基培养静止后 ,分别加入血小板源性生长因子 (PDGF) ( 2 0ng ml)、PDGF +PD980 5 9( 2 0ng ml +2 0mmol L)、PDGF +硫酸新霉素 ( 2 0ng ml+10mmol L)等刺激因素 ,以无血清培养基培养的VSMC作对照。在刺激后 90min收集细胞 ,用免疫沉淀法检测MAPK(p44 p42 )活性的改变。在刺激后 6和 2 4h收集细胞 ,用Western蛋白印迹法检测p2 7、p5 7和p2 1蛋白表达量。结果 PDGF刺激后 ,MAPK活性明显升高 (较对照组增加 46 6 % ) ,同时VSMC明显增殖。刺激 2 4h后 ,细胞增殖程度为对照组的 1 4 3倍 ,p2 7蛋白的表达量显著下降至对照组的 71% ;p2 1和p5 7蛋白表达量却明显增加 ;加用PD980 5 8(MAPK抑制剂 )和新霉素 (PLC抑制剂 )可明显抑制PDGF引起的上述改变 ,MAPK活性分别较PDGF刺激组下降了 46 %和 37% ,p2 7蛋白表达量则分别为PDGF刺激组的 1 77倍和 1 4 9倍 ,细胞增殖程度分别降为后者的6 8%和 6 5 %。结论 MAPK(p44 4 2 )变化的幅度是决定CKI表达量和VSMC?

Objective Our previous study showed that there were different changes of cyclin dependent kinase inhibitors (CKIs) in vascular smooth muscle cells stimulated by PDGF and suggested that p27 was a key factor in transduction pathway of vascular smooth muscle cells proliferation. The purpose of this study was to elucidate the effects of mitogen-activated protein kinase (MAPK) pathway and phospholipase C pathway on regulation of CKIs protein expression and vascular smooth muscle cells proliferation. Methods Sprague Dawley male rat aorta media smooth muscle cells(VSMC) were isolated and cultured. After serum withdrawal for 48 hours to induce quiescence, PDGF(20 ng/ml) , PDGF+PD98059(20 ng/ml+20 mmol/L) and PDGF+Neomycin(20 ng/ml+10 mmol/L) were added into serum-free medium, respectively. VSMCs were harvested at indicated time after stimulation. Protein levels of p27, p21 and p57 were investigated with Western blot analysis. The MAPK was evaluated by immunoprecipitation test after stimulated for 90 minutes. Results MAPK activity was enhanced by 4.66 folds by PDGF stimulation, meanwhile the expression of p27 protein was decreased to 71% of the control. Proliferation of vascular smooth muscle cells was observed .The expression of p27 protein was inhibited with co-treatment of PD98059 or Neomycin as compared to PDGF group,while the proliferation of vascular smooth muscle cells was decreased. Conclusion MAPK (Erk) pathway is an important signalling pathway of extracellular proliferation signal conducting to nucleus and expression of CKIs in VSMC. Phospholipase C pathway is involved in signalling of PDGF in VSMC proliferation.