【作者】 郑艳；

【导师】 黄玮；

【作者基本信息】 重庆医科大学 ， 内科学， 2021， 硕士

【摘要】 目的:肺动脉高压(Pulmonary arterial hypertension,PAH)是较为罕见的肺血管性疾病,主要表现为肺动脉压力升高,其病理特征以血管的重构为主。肺动脉平滑肌细胞由收缩表型向合成表型转化是肺血管重构的重要原因之一。成纤维细胞生长因子9(Fibroblast growth factor 9,FGF9)是一种自分泌或旁分泌细胞因子,参与肿瘤生长、胚胎发育、炎症及血管生成等病理生理过程。本研究主要探讨FGF9是否通过调节肺动脉平滑肌细胞(Pulmonary artery smooth muscle cells,PASMCs)表型转化而在肺动脉高压的病理过程中发挥作用。方法:本研究采用野百合碱(Monocrotaline,MCT)诱导的PAH大鼠模型,Western blot检测PAH大鼠中FGF9的表达。培养原代大鼠PASMCs,血小板衍生生长因子(Platelet-derived growth factor BB,PDGF-BB)和低氧(Hypoxia)诱导PASMCs由收缩表型向合成表型转化,Western blot检测PASMCs中FGF9的表达。在病理情况下添加外源性重组人FGF9(Recombinant human fibroblast growth factor 9,rh FGF9)蛋白干预细胞,Western blot检测细胞中收缩表型标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)与合成表型标志物骨桥蛋白(Osteopontin,OPN)及血小板衍生生长因子受体β(Platelet-derived growth factor receptorβ,PDGFR-β)的表达,划痕实验检测rh FGF9对细胞迁移能力的影响。si RNA抑制FGF9,Western blot检测FGF9蛋白、收缩表型标志物α-SMA蛋白及合成表型标志物OPN蛋白的表达。结果:(1)Western blot结果显示,在MCT-PAH中,与Control组相比,MCT组中FGF9蛋白的表达下降(p<0.001)。(2)在PASMCs实验中,与Control组相比,PDGF-BB组和低氧组中FGF9蛋白的表达显著降低(p<0.001)。(3)外源性重组人FGF9蛋白干预细胞后,划痕结果显示,rh FGF9明显抑制了PDGF-BB促进PASMCs迁移的作用;Western blot结果显示,与Control组相比,PDGF-BB组和低氧组的细胞明显由收缩表型向合成表型转化,α-SMA的表达降低(p<0.05)伴OPN的表达升高(p<0.05),同时PDGFR-β的表达降低(p<0.05);与PDGF-BB组及低氧组相比,rh FGF9+PDGF-BB组与rh FGF9+低氧组中α-SMA的表达明显升高(p<0.05)伴OPN的表达降低(p<0.05),且rh FGF9+PDGF-BB组中PDGFR-β的表达无显著变化,差异无统计学意义,提示rh FGF9抑制了PDGF-BB和低氧诱导的细胞表型转化作用。(4)下调FGF9后,PASMCs中α-SMA表达显著降低(p<0.05),而OPN的表达无显著改变,差异无统计学意义。结论:FGF9抑制PDGF-BB和低氧诱导的肺动脉平滑肌细胞表型转化,并且FGF9不影响PDGF-BB对其受体PDGFR-β的改变,同时FGF9的下调改变细胞的收缩表型,提示FGF9参与平滑肌细胞的表型转化,表明FGF9与肺动脉高压的发生密切相关。更多还原

【Abstract】 Objective: Pulmonary arterial hypertension(PAH)is a relatively rare pulmonary vascular disease,which is mainly manifested by increased pulmonary artery pressure,and its pathological characteristics are mainly vascular remodeling.The transformation of pulmonary artery smooth muscle cells from contractile phenotype to synthetic phenotype is one of the important factors for pulmonary vascular remodeling.Fibroblast growth factor 9 is an autocrine or paracrine cytokine involved in pathophysiological processes such as tumor growth,embryonic development,inflammation and angiogenesis.This study mainly explored whether FGF9 played a role in the pathological process of pulmonary hypertension by regulating the phenotypic transformation of pulmonary artery smooth muscle cells(PASMCs).Methods: Animal models of pulmonary arterial hypertension induced by monocrotaline(MCT),and the expression of FGF9 in the PAH model was detected by Western blot.Rat primary PASMCs were cultured in vitro,platelet-derived growth factor BB(PDGF-BB)and hypoxia induced phenotypic transformation of pulmonary artery smooth muscle cells,the expression of FGF9 and shrinkage phenotype marker α-smooth muscle actin(α-SMA)were detected by Western blot.Exogenous recombinant human FGF9(rhFGF9)interfered with cells under pathological stimulation,the expression of shrinkage phenotype marker α-SMA,Synthetic phenotypic marker osteopontin(OPN),and platelet-derived growth factor receptor β(PDGFR-β)were detected by Western blot,and the effect of rhFGF9 on cell migration was detected by the scratch assay.Specifically knock down FGF9,the expression of FGF9 and α-SMA and OPN were detected by Western blot.Results:(1)Compared with the Control group,Western blot results showed that the expression of FGF9 protein in MCT decreased(p<0.05).(2)In PASMCs study,the expression of FGF9 protein in the PDGF-BB group and hypoxia group were significantly reduced compared with the Control group(p<0.05).(3)After treated with rhFGF9 in PASMCs,in the cell scratch assay,rhFGF9 significantly inhibited the migration of PASMCs induced by PDGF-BB.Western blot results showed that compared with the Control group,cells transform from contractile phenotype to synthetic phenotype in the PDGF-BB group and hypoxia group,the expression ofα-SMA decreased(p<0.05),accompanied by up-regulation of synthetic phenotypic marker OPN(p<0.05),and the expression of PDGFR-βdecreased(p<0.05);compared with PDGF-BB group and hypoxia group,in the rhFGF9+ PDGF-BB group and rhFGF9+hypoxia group,the expression of α-SMA was significantly increased(p<0.05)and the expression of OPN was decreased(p<0.05),and the expression of PDGFR-β in rhFGF9+PDGF-BB group did not change significantly,the difference was not statistically significant,this suggesting that rhFGF9 inhibited PDGF-BB and hypoxia-induced cell phenotypic transformation.(4)After knocking down FGF9,the expression of α-SMA protein was significantly reduced(p<0.05),while the expression of OPN did not change significantly,the difference was not statistically significantConclusions: FGF9 inhibits phenotypic transformation and migration induced by PDGF-BB and hypoxia of pulmonary artery smooth muscle cells,without affecting PDGF-BB activated receptor PDGFR-β,at the same time,the down-regulation of FGF9 affected the contractile phenotype of cells.FGF9 is involved in the phenotypic transformation of PASMCs,FGF9 is closely related to the occurrence of pulmonary hypertension.更多还原