【作者】 杨群；

【导师】 赵建平；

【作者基本信息】 华中科技大学 ， 内科学， 2017， 硕士

【摘要】 目的:体外研究糖皮质激素对TGF-β1所致肺纤维化的影响。方法:本研究主要通过转化生长因子β1（TGF-β1）诱导肺泡II型上皮细胞系A549发生上皮间质转化（EMT）构建肺纤维化模型,通过real-time PCR方法筛选适宜的TGF-β1浓度,同时观察该浓度作用下细胞形态学变化,并采用Western blot检测相关蛋白的表达。然后分别于TGF-β1刺激A549 24h或48h后予以10^-8M、10^-7M、10^-6M、10^-5M、、10^-4M等浓度地塞米松（Dex）干预,采用Western blot检测E-cadherin（E-cad）、α-SMA、fibronectin的表达情况。结果:2ng/ml TGF-β1可以使A549发生形态学改变,通过real-time PCR检测发现TGF-β1刺激A549后,上皮细胞标志物E-cad mRNA表达下降、间质细胞标志物Collagen I mRNA和Vimentin mRNA表达增加,α-SMA mRNA呈降低表现;显微镜下观察细胞形态由原来的鹅卵石样变成纺锤体样,细胞间连接减少,且随着时间的延长细胞形态学改变越明显;Western blot检测发现上皮细胞标志物E-cad表达下降、间质细胞标志物fibronectin表达增高,α-SMA无明显变化。而予以不同浓度Dex干预对TGF-β1所致上皮细胞E-cad的降低无明显改善,且低浓度情况下有促进fibronectin表达的作用,较早予以高浓度Dex可能会减轻fibronectin的表达。结论:2ng/ml TGF-β1可以诱导A549产生EMT,且随着时间的延长细胞形态及蛋白表达差异越明显。而不同浓度Dex对TGF-β1所致肺纤维无明显改善作用,且低浓度的Dex能促进EMT的加重,较早予以高浓度Dex可能会减轻EMT细胞外基质的产生。更多还原

【Abstract】 Objective: The epithelial-to-mesenchymal transition(EMT)is one of the key mechanisms involved in pulmonary fibrosis.Glucocorticoid,one of the most commonly used anti-inflammatory drugs,inhibits the deposition of extracellular matrix in-dependent of its anti-inflammatory effect.In the study,we assessed the effects of glucocorticoid on TGF-β1-induced pulmonary fibrosis in the human alveolar epithelial cell line(A549).Methods: A549 cells were examined for evidence and optimum concentration of EMT after treatment with TGF-β1.EMT was assessed by: morphology under phase-contrast microscopy;mRNA transcripts for E-cadherin(E-cad),vimentin,a-SMA and Collagen I were analyzed by real-time PCR;The protein expression for E-cad,α-SMA and fibronectin were analyzed by Western blot.Then A549 cells were exposed to different concentrations of dexamethasone(Dex)by stimulation with TGF-β1.Western analysis of cell lysates for expression of epithelial phenotypic markers including E-cad,expression of mesenchymal phenotypic markers including α-SMA and fibronectin.Results: The data showed that TGF-β1 induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in the concentration as low as 2ng/ml and a time-dependent manner.The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers fibronectin and vimentin,concomitant with a downregulation of the epithelial phenotype marker E-cad.Dex could not increased the expression of epithelial phenotypic marker E-cad,and could increase mesenchymal phenotypic marker fibronectin in A549 cells,especially in the low concentration.The high concentration of Dex might reduce the generation of fibronectin in the early stages of EMT.Conclusion: TGF-β1 could induce A549 cells undergo pulmonary fibrosis in the concentration as low as 2ng/ml and a time-dependent manner.However,different concentrations of Dex did not have no significant effects on the TGF-β1-induced pulmonary fibrosis,the low concentrations of Dex could promote the deterioration of pulmonary fibrosis,and high concentration of Dex could reduce the generation of extracellular matrix in the early stages of pulmonary fibrosis.更多还原