磷脂酰肌醇3激酶通路对大鼠肺动脉平滑肌细胞分化状态和增殖活性的调控作用  

Role of phosphatidyl inositol 3-kinase pathway in regulating dedifferentiation and proliferation of rat pulmonary arterial vascular smooth muscle cells

作　　者：范志宇[1] 李春雨 秦超毅 谢亮[3] 王小双[4] 高正祥[5] 强巴措珍[1] 王涛[1] 余莉[1] 刘瀚旻[1] 

机构地区：[1]四川大学华西第二医院儿科,成都610041 [2]西部妇幼医学研究院肺血管重构研究室 [3]妇儿疾病与出生缺陷教育部重点实验室 [4]绵阳市中心医院儿科 [5]攀枝花市中心医院儿科

出　　处：《中华实用儿科临床杂志》2013年第18期1417-1421,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家重点基础研究发展计划项目（2007CB511905）;教育部创新团队项目（IRT0935）

摘　　要：目的 研究磷脂酰肌醇3激酶（PI3K）通路对大鼠肺动脉平滑肌细胞（PASMC）分化状态和增殖活性的调控作用.方法 体外培养雄性SD大鼠PASMC并传至第3代用于实验,将细胞分为8组：空白对照组,血小板源性生长因子（PDGF）诱导组,二甲基亚砜（DMSO）溶剂对照组,LY294002干预组,小分子干扰RNA（siRNA）干预组,Control-siRNA对照组,空病毒对照组,重组腺病毒诱导组.用实时荧光定量PCR（RT-qPCR）检测重组腺病毒及siRNA转染的PASMC中PI3K α调节亚基（PIK3CA）的表达情况,用Western blot检测平滑肌α肌动蛋白（α-SM actin）的表达水平,用RT-qPCR检测α-SM actin mRNA水平,用四甲基偶氮唑盐（MMT）比色实验及噻唑盐细胞计数-8（CCK-8）试剂盒检测PASMC的增殖活性.结果 重组腺病毒诱导组PASMC中PIK3CA mRNA水平高于空白对照组（P＜0.05）,siRNA干预组中PIK3CA mRNA水平低于空白对照组（P＜0.05）.与空白对照组相比,PDGF诱导组α-SM actin的mRNA水平和蛋白表达水平明显降低（P均＜0.05）,增殖活性增高（P＜0.05）,LY294002可阻断PDGF-BB诱导的α-SM actin的基因转录、蛋白表达及PASMC增殖（P＜0.05）,予PIK3 CA-siRNA干预也可抑制PDGF-BB诱导的α-SM actin mRNA水平、蛋白表达水平及细胞增殖活性（P均＜0.05）；重组腺病毒诱导组α-SM actin的mRNA水平和蛋白表达水平与空白对照组相比也明显降低（P均＜0.05）,增殖活性增高（P＜0.05）.结论 在肺血管重构过程中,PI3K通路对大鼠PASMC去分化和细胞增殖具有正向调控作用.Objective To investigate the role of phosphatidyl inositol 3-kinase （PI3K） in rat pulmonary arterial vascular smooth muscle cell （PASMC） dedifferentiation and proliferation.Methods PASMC were isolated from Sprague-Dawley（SD） rat arteriae pulmonalis and subcultured to the third generation for experiment.PASMC were divided into blank group,platelet derived growth factor（PDGF） group,dimethylsul foxide（DMSO） group,LY294002 group,small interfening RNA （siRNA） group,control-siRNA group,empty-virus group,recombinant-adenovirus group.Real-time fluorescence quantitative polymerase chain reaction（RT-qPCR） was performed to detect the PIK3CA mRNA level in PASMC transfected with pAdxsi-GFP-PIK3CA recombinant adenovirus and PIK3CA-siRNA.Western blot analysis was used to measure the expression level of alpha smooth muscle actin（α-SM actin）.The mRNA level of α-SM actin was detected by RT-qPCR.The methyl thiazolyl tetrazolium（MTT） assay and thiazole cell counting kit-8 （CCK-8）method were used to assess proliferation activity of PASMC.Results The PIK3CA mRNA level was significantly increased in recombinant-adenovirus group compared with blank group（P ＜0.05）.The mRNA level of PIK3CA in siRNA group was lower than that in blank group（P ＜ 0.05）.Compared with blank group,significantly lower α-SM actin mRNA and protein levels and a significantly higher proliferation activity were detected in PDGF group（all P ＜ 0.05）.PI3K inhibitor LY294002 blocked this down-regulation of α-SM actin gene expression and up-regulation of proliferation activity induced by PDGF-BB（P ＜0.05）.PIK3CA-siRNA also attenuated these biological effects.The α-SM actin mRNA level and protein expression level in recombinant-adenovirus group were lower than those in blank group （P ＜ 0.05）,while the proliferation activity in the former group was greater（P ＜ 0.05）.Conclusions PI3 K pathway plays a vital role in vascular remodeling through a positive regulation of dedifferentiation and prolifera

关 键 词：磷脂酰肌醇3激酶 肺动脉平滑肌细胞 去分化 增殖 

分 类 号：R363[医药卫生—病理学]