血管紧张素Ⅱ通过Raf/MEK/ERK通路对PDGF诱导的气道上皮细胞转分化的影响  

Effects of angiotensinⅡon PDGF induced trans-differentiation of airway epithelial cells via Raf/MEK/ERK pathway

作　　者：杨丹芬[1] 谢圆媛[1] 康睿[2] YANG Danfen;XIE Yuanyuan;KANG Rui(Department of Geriatrics,Affiliated Hospital of Yan’an University,Shaanxi,Yan’an 716000,China;不详)

机构地区：[1]延安大学附属医院老年病科,陕西省延安市716000 [2]延安大学附属医院呼吸内科,陕西省延安市716000

出　　处：《河北医药》2022年第8期1141-1146,共6页Hebei Medical Journal

摘　　要：目的探讨血管紧张素Ⅱ(AngⅡ)对加速纤维肉瘤蛋白(Raf)/丝裂原活化的细胞外信号调节激酶(MEK)/细胞外信号调节激酶(ERK)信号通路的调控作用,及对血小板源生长因子(PDGF)诱导的气道上皮细胞转分化(EMT)的影响。方法取人气道上皮细胞株16-HBE,分为对照组、PDGF不同浓度刺激组(5、25、50、100、200 ng/L),用免疫荧光法检测各组细胞EMT标志物波形蛋白(vimentin)阳性表达,选出合适的PDGF诱导浓度。将16-HBE细胞分为正常对照组、PDGF诱导组(100 ng/L)、AngⅡ不同浓度组(10^(-7)、10^(-6)、10^(-5) mol/L),用倒置显微镜检测细胞形态;蛋白免疫印迹法(Western blot)检测细胞EMT相关蛋白E-粘钙素(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、vimentin,气道纤维化相关蛋白-纤维连接蛋白(FN)、胶原蛋白Ⅳ(ColⅣ),Raf/MEK/ERK通路相关蛋白Raf、MEK及磷酸化水平(p-MEK)、ERK及磷酸化水平(p-ERK)、转化生长因子β1(TGF-β1)等蛋白相对表达水平。将16-HBE细胞分为正常对照组、PDGF诱导组、AngⅡ组(10^(-6) mol/L)、Raf/MEK/ERK通路抑制剂(sorafenib)组(10μmoL/L)、AngⅡ+sorafenib(10^(-6) mol/L+10μmol/L)组,检测细胞形态及Raf/MEK/ERK通路相关蛋白表达。结果PDGF不同浓度处理下,细胞vimentin阳性表达随浓度升高逐渐升高,并在100~200 ng/L时上升至稳定水平,选取100 ng/L为诱导浓度。AngⅡ不同浓度处理后,与正常对照组比较,PDGF诱导组细胞紧密连接减少,细胞呈长梭形等改变,E-cadherin表达降低(P<0.05),α-SMA、vimentin、FN、ColⅣ、Raf、p-MEK/MEK、p-ERK/ERK、TGF-β1蛋白表达升高(P<0.05);与PDGF诱导组比较,AngⅡ不同浓度处理组细胞长梭形改变进一步加重,E-cadherin表达进一步降低(P<0.05),α-SMA、vimentin、FN、ColⅣ、Raf、p-MEK/MEK、p-ERK/ERK、TGF-β1蛋白表达进一步升高(P<0.05),且AngⅡ浓度10^(-6) mol/L组及10^(-5)mol/L浓度组上述指标变化优于10^(-7)mol/L组,AngⅡ浓度10^(-6) mol/L组与10^(-5)mol/LObjective To investigate the regulatory effect of angiotensinⅡ(AngⅡ)on rapidly accelerating fibrosarcoma protein(Raf)/mitogen activatory extracellular signal regulated kinase(MEK)/extracellular signal regulated kinase(ERK)signaling pathway,and its effects on the trans-differentiation(EMT)of airway epithelial cells induced by platelet derived growth factor(PDGF).Methods Human airway epithelial cell line 16 HBE was divided into control group and PDGF stimulation groups with different concentrions(5,25,50,100,200ng/L).The positive expression levels of EMT landmark vimentin of cells were detected by immunofluorescence.The 16 HBE cells were divided into normal control group,PDGF induction group(100ng/L)and AngⅡgroups with different concentrations(10^(-7),10^(-6),10^(-5) mol/L).The cell morphology was detected by inverted microscope.The protein relative expression levels of EMT related proteins E cadherin(E cadherin),αsmooth muscle actin(α-SMA),vimentin,airway fibrosis associated proteins fibronectin(FN),collagenⅣ(ColⅣ),Raf/MEK/ERK pathway related proteins Raf,MEK and phosphorylated MEK(p-MEK),ERK and phosphorylated ERK(p-ERK)and transforming growth factor beta 1(TGF-β1)were detected.Moreover the 16 HBE cells were divided into normal control group,PDGF induction group,AngⅡgroup(10^(-6) mol/L),Raf/MEK/ERK pathway inhibitor(sorafenib)group(10μmol/L),AngⅡ+sorafenib(10^(-6) mol/L+10μmol/L)group.The cell morphology and expression levels of Raf/MEK/ERK pathway related proteins were observed.Results The positive expression levels of vimentin were increased with the increase of PDGF concentration,which reached a stable level at the concentration of 100~200ng/L.After treatment by different concentrations of AngⅡ,the tight junction of cells in PDGF induced group was significantly decreased,the cells showed spindle changes and the expression levels of E cadherin were significantly decreased(P<0.05),however the protein expressions ofαSMA,vimentin,FN,ColⅣ,Raf,p-MEK/MEK,p-ERK/ERK and TGF-β1 were significantl

关 键 词：血管紧张素Ⅱ 气道上皮细胞 加速纤维肉瘤蛋白 丝裂原活化的细胞外信号调节激酶 细胞外信号调节激酶 血小板源生长因子 转分化 

分 类 号：R562.25[医药卫生—呼吸系统]