PDGF-BB通过SIRT3影响糖酵解途径调控肺动脉平滑肌细胞的表型转化  

Platelet derived growth factor-BB regulates phenotype transformation of pulmonary artery smooth muscle cells via SIRT3 affecting glycolytic pathway

作　　者：卢毅[1] 陈蕊[1] 马金玉 王伦平 仇苓苓[1] 王翠平[1] 严金川[1] 刘培晶[1] Lu Yi;Chen Rui;Ma Jinyu;Wang Lunping;Qiu Lingling;Wang Cuiping;Yan Jinchuan;Liu Peijing(Department of Cardiology,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,China)

机构地区：[1]江苏大学附属医院心内科,镇江212001

出　　处：《中华心血管病杂志》2019年第12期993-999,共7页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81400208);江苏省自然基金(BK20181227);镇江市社会发展基金(SH2018043)。

摘　　要：目的探讨血小板衍生生长因子(PDGF)-BB是否通过去乙酰化酶沉默信息调节因子3(SIRT3)影响糖酵解途径进而调控肺动脉平滑肌细胞(PASMC)的表型转化。方法采用组织贴块法培养大鼠原代PASMC。分组实验中以PDGF-BB作为刺激因子,以2-脱氧葡萄糖(2-DG)作为糖酵解途径抑制剂,将PASMC分为空白对照组、PDGF-BB(30 ng/ml)组、PDGF-BB(30 ng/ml)+2-DG(10 mmol/L)组;在慢病毒过表达实验中,将PASMC分为空白对照组、PDGF-BB(30 ng/ml)组,PDGF-BB(30 ng/ml)+SIRT3过表达组,PDGF-BB(30 ng/ml)+空载体组。分别采用实时定量PCR(RT-qPCR)及Western blot法检测PASMC中α-平滑肌肌动蛋白(α-SMA)、平滑肌肌球蛋白重链(SM-MHC)、钙调蛋白(calponin)、波形蛋白(vimentin)等表型指标的表达。同时采用细胞免疫荧光染色检测α-SMA表达水平。采用5-乙炔基-2′-脱氧尿苷(EDU)检测PASMC增殖情况。采用Western blot法检测PDGF-BB刺激后SIRT3的蛋白表达。在慢病毒过表达实验中,分别采用RT-qPCR、Western blot法检测葡萄糖转运蛋白1(Glut1)及有氧糖酵解酶mRNA和蛋白的表达水平。结果(1)PDGF-BB通过糖酵解途径影响PASMC表型转化:PDGF-BB组细胞收缩表型指标SM-MHC、calponin、α-SMA的mRNA和蛋白表达水平均低于空白对照组(P均<0.05),合成表型指标vimentin的mRNA和蛋白表达水平则均高于空白对照组(P均<0.05)。PDGF-BB+2-DG组细胞收缩表型指标SM-MHC、calponin、α-SMA的mRNA和蛋白表达水平均高于PDGF-BB组(P均<0.05),合成表型指标vimentin的mRNA和蛋白表达水平则均低于PDGF-BB组(P均<0.05)。免疫荧光结果显示,PDGF-BB组α-SMA阳性细胞数少于空白对照组,而PDGF-BB+2-DG组则多于PDGF-BB组。(2)PDGF-BB通过糖酵解促进细胞增殖:PDGF-BB组细胞增殖数多于空白对照组,而PDGF-BB+2-DG组则少于PDGF-BB组。(3)PDGF-BB抑制PASMC的SIRT3蛋白表达:PDGF-BB组细胞SIRT3蛋白表达水平低于空白对照组(P<0.05)。(4)PDGF-BB通过SIRT3影响糖酵解:RTTo investigate whether platelet-derived growth factor-BB(PDGF-BB)can regulate phenotypic transformation of pulmonary artery smooth muscle cells(PASMCs)via SIRT3 affecting glycolytic pathway.Methods The PASMCs were isolated from Sprague Dawley rats.PASMCs were divided into 3 groups by using 2-deoxyglucose(2-DG),an inhibitor of the glycolytic pathway:normal control group,PDGF-BB group(30 ng/ml)and PDGF-BB(30 ng/ml)+2-DG(10 mmol/L)group.In lentivirus-mediated overexpression assay,cells were divided into control group,PDGF-BB group(30 ng/ml),PDGF-BB+deacetylase sirtuin-3(SIRT3)overexpression group and PDGF-BB+empty vector group.The expression levels of phenotype related index such asα-smooth muscle actin(α-SMA),smooth muscle myosin heavy chain(SM-MHC),calponin,vimentin were detected by qRT-PCR and Western blot.Meanwhile,the expression ofα-SMA was detected by cellular immunofluorescence staining.EDU staining was used to detect the proliferation of PASMCs.The expression of SIRT3 was detected by Western blot.The expressions of glucose transporter 1 and aerobic glycolytic enzymes were detected by qRT-PCR and Western blot in lentivirus-mediated overexpression assay.Results(1)PDGF-BB affects PASMCs phenotypic transformation through glycolytic pathway:compared with normal control group,PDGF-BB significantly decreased the expressions of contractile phenotype markers such asα-SMA,SM-MHC,calponin mRNA and protein(all P<0.05),but it increased the expressions of the synthetic phenotype marker vimentin mRNA and protein(both P<0.05).Cellular immunofluorescence assay showed that PDGF-BB significantly decreased the number ofα-SMA positive cells,while 2-DG reversed the process.(2)PDGF-BB promoted cell proliferation through glycolytic pathway:the proliferation of PASMCs was significantly higher in PDGF-BB group than in control group(P<0.05),and which could be significantly reduced by 2-DG(P<0.05).(3)PDGF-BB inhibited the expression of SIRT3 protein in PASMCs:the expression of SIRT3 protein in PDGF-BB group was lower than that in

关 键 词：肺动脉 肌细胞 平滑肌 糖酵解 血小板衍生生长因子 SIRT3 

分 类 号：R544[医药卫生—心血管疾病]