摘要
目的建立PDGF-BB诱导的原代大鼠肺动脉平滑肌细胞（pulmonary vascular smooth musclecells，PASMCs）的增殖细胞模型，通过AMPK激动剂AICAR干预，探讨AICAR对PASMCs细胞周期和增殖的影响及其机制，为肺血管重构防治寻找靶点。方法通过20ng／mlPDGF-BB刺激诱导PASMCs增殖建立细胞模型，采用AICAR（0．5mmol／L）干预PDGF-BB诱导的PASMCs增殖，Westernblot法检测总的和磷酸化的AMPK，CCK-8检测PASMCs增殖，流式细胞仪分析细胞周期，实时定量PCR（RT-PCR）检测cyclinDl、cyclinE、CDK2／4／6mRNA的表达。结果Westernblot法检测表明AICAR可以活化AMPK，CCK-8检测结果表明AICAR能够抑制PDGF-BB诱导的PASMCs增殖；流式细胞仪检测结果表明AICAR能够抑制细胞周期于G0／G1期，RT-PCR结果表明AICAR可以抑制cyclinDl、cyclinE、CDK2、CDK4和CDK6的mRNA的表达。结论AICAR通过抑制cy-clinDl、cyclinE、CDK2／4／6的mRNA表达阻滞细胞周期于G0／G1-S期，抑制PASMCs增殖。
Objective To establish the PDGF -BB -induced proliferation of rat pulmonary artery smooth muscle cells（PAbMGs） , and investigate the effects of the AMPK agonist AICAR on the cell cycle of PASMCs, in order to search new drugs for prevention of pulmo- nary vascular remodeling. Methods 20ng/ml PDGF - BB was used to induced the proliferation of PASMCs, and the effect of 0.5mmol/ L AICAR on the proliferation of PASMCs was observed. Western blot was used to detect the total and phosphorylated AMPK. The prolifer- ation of PASMCs was determined by CCK - 8. The mRNA expression of cyclinD1, cyclinE and CDK2/4/6 were detected by flow cytome- try analysis cell cycle, quantitative real - time PCR. Results Western blot results indicated AICAR could promote the activation of AMPK. CCK - 8 test results showed that AICAR blocked the proliferation of PASMCs induced by PDGF - BB. Flow cytometry analysis in- dicated that AICAR arrested the cell cycle in G0/G1 to S phase. RT - PCR results demonstrated that AICAR inhibited the mRNA expres- sion of cyclinD1, cyclinE and CDK2/4/6. Conclusion The AMPK agonist AICAR can block the proliferation of PASMCs induced through arrest cell cycle in Go/G1 - S phase by regulation the mRNA expression of cyclin D1, cyclinE, CDK2/4/6, and AICAR has a po- tential application in preventing pulmonary vascular remodeling.
出处
《医学研究杂志》
2015年第5期111-113,共3页
Journal of Medical Research
