16HBE细胞上皮间质转化模型建立以及麻黄-甘草药对对其影响
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摘要
目的体外诱导人支气管上皮细胞(16HBE)上皮间质转化(EMT)最适条件摸索以及探讨麻黄-甘草对EMT的影响。方法以16HBE细胞株为研究对象,分别给予转化生长因子β1(TGF-β1)单一刺激以及血清饥饿联合刺激,运用Western blot法检测上皮及间质特征性蛋白的表达。运用免疫荧光检测E-Cadherin和N-Cadherin胞内表达分布情况。运用qPCR检测上皮及间质特征性基因的表达水平。血清饥饿联合TGF-β1刺激,给予麻黄-甘草药对干预,Western blot法检测上皮及间质特征性蛋白的表达水平。结果 TGF-β1可诱导16HBE细胞由结构规则、质密均匀的鹅卵石状向纺锤体状转化。TGF-β1单独刺激可诱导16HBE细胞N-Cadherin、α-SMA蛋白表达升高,对E-Cadherin、ZO-1蛋白表达未见明显影响。而当细胞密度为15%时,血清饥饿联合TGF-β1可诱导16HBE细胞N-Cadherin、α-SMA、Vimentin、CollagenⅠ蛋白、基因水平升高以及E-Cadherin、ZO-1蛋白、基因水平降低,并且E-Cadherin蛋白分布紊乱,上皮结构破坏。血清饥饿联合TGF-β1刺激,麻黄-甘草药对可抑制N-Cadherin、α-SMA的表达,并恢复E-Cadherin蛋白的表达,对ZO-1无明显影响。结论血清饥饿联合TGF-β1能体外诱导16HBE细胞建立稳定可重复的EMT模型,可用于体外研究哮喘发生EMT相关机制。麻黄-甘草药对一定程度上可抑制16HBE细胞EMT过程。
OBJECTIVE To establish the epithelial-mesenchymal transformation(EMT) in 16 HBE cells and to investigate the effects of couplet medicines on EMT in 16 HBE cells. METHODS Western blot was applied to detect the mark proteins expression in 16 HBE cells treated with TGF-β1 or combined with serum starvation. The distribution of E-Cadherin and N-Cadherin were detected by immunofluorescence. The qPCR was used for detecting the mark mRNA levels. The effects of couplet medicines on the expression of EMT associated proteins were detected by Western blot. RESULTS TGF-β1 induced 16 HBE cells type changed, from a cobblestone to a spindle. TGF-β1 increased the expression of N-Cadherin and α-SMA, but not affected the expression of E-Cadherin and ZO-1. Meanwhile, the protein and mRNA expression of N-Cadherin, α-SMA, Vimentin, Collagen Ⅰ were significantly increased in 16 HBE cells treated with serum starvation and TGF-β1. At the same time, the protein and mRNA levels of E-Cadherin and ZO-1 were obviously decreased. The distribution of E-Cadherin was disorder and the epithelial structure was damaged. Compared with the stimulate group, the couplet medicines decreased the expression of N-Cadherin and α-SMA, while increased the expression of E-Cadherin. CONCLUSION 16 HBE cells, stimulated with serum starvation and TGF-β1, undergo the EMT process, which can be applied to study the mechanism of EMT in asthma. The Mahuang-Gancao couplet medicines can inhibit EMT in 16 HBE cells.
OBJECTIVE To establish the epithelial-mesenchymal transformation(EMT) in 16 HBE cells and to investigate the effects of couplet medicines on EMT in 16 HBE cells. METHODS Western blot was applied to detect the mark proteins expression in 16 HBE cells treated with TGF-β1 or combined with serum starvation. The distribution of E-Cadherin and N-Cadherin were detected by immunofluorescence. The qPCR was used for detecting the mark mRNA levels. The effects of couplet medicines on the expression of EMT associated proteins were detected by Western blot. RESULTS TGF-β1 induced 16 HBE cells type changed, from a cobblestone to a spindle. TGF-β1 increased the expression of N-Cadherin and α-SMA, but not affected the expression of E-Cadherin and ZO-1. Meanwhile, the protein and mRNA expression of N-Cadherin, α-SMA, Vimentin, Collagen Ⅰ were significantly increased in 16 HBE cells treated with serum starvation and TGF-β1. At the same time, the protein and mRNA levels of E-Cadherin and ZO-1 were obviously decreased. The distribution of E-Cadherin was disorder and the epithelial structure was damaged. Compared with the stimulate group, the couplet medicines decreased the expression of N-Cadherin and α-SMA, while increased the expression of E-Cadherin. CONCLUSION 16 HBE cells, stimulated with serum starvation and TGF-β1, undergo the EMT process, which can be applied to study the mechanism of EMT in asthma. The Mahuang-Gancao couplet medicines can inhibit EMT in 16 HBE cells.
引文
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[14] 季律,魏盼,王璨,等.麻黄-甘草药对对脂多糖诱导的RAW264.7细胞的体外抗炎作用分析[J].中国实验方剂学杂志,2017,23(18):83-88.
[15] 魏盼,季律,袁为远,等.麻黄-甘草药对对小鼠脾细胞的增殖以及Th1/Th2平衡的影响[J].南京中医药大学学报,2018,34(4):371-375,380.
[2] QUIRT J,HILDEBRAND KJ,MAZZA J,et al.Allergy,asthma,and clinical immunology [J].Asthma,2018,14(2):50.
[3] GANESAN S,SAJJAN US.Repair and remodeling of airway epithelium after injury in chronic obstructive pulmonary disease [J].Curr Respir Care Rep,2013,2(3),145-154.
[4] FATEMEH M,HOLIS MR,JENNIFER C.Disruption of β-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair [J].Int J Biochem Cell Biol,2015,68:59-69.
[5] THEVENOT PT,SARAVIA J,JIN N,et al.Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells [J].Am J Resp Cell Mol Biol,2013,48(2):188-197.
[6] 唐于平,束晓云,李伟霞,等.药对研究(Ⅰ)——药对的形成与发展[J].中国中药杂志,2013,38(24):4185-4190.
[7] 郝知音.中药麻黄汤加减方治疗急性喘息型支气管炎的临床效果探讨[J].世界最新医学信息文摘,2019,19(7):154-157.
[8] 曹登瑞.小青龙汤对慢性阻塞性肺病气道炎症及气道重塑的影响[J].西部医学,2009,21(4):575-577.
[9] 李恋曲,季律,魏盼,等.麻黄-甘草药对抑制Th2型变应性接触性皮炎的作用及机制探讨[J].南京中医药大学学报,2018,34(3):282-286.
[10] 季律.黄芪-防风/麻黄-甘草“药对”防治过敏性疾病机制探索[D].南京:南京中医药大学,2017.
[11] JOHNSON JR,ROOS A,BERG T,et al.Chronic respiratory aeroallergen exposure in mice induces epithelial-mesenchymal transition in the large airways [J].PLoS ONE,2011,6(1):e16175.
[12] 刘婷,刘丹,陈葳,等.4种卵巢癌细胞缺氧模型的比较[J].中国妇幼健康研究,2013,24(4):511-513.
[13] REYAZ UR,DEBASIS N,SOUNEEK C,et al.Differential regulation of NM23-H1 under hypoxic and serum starvation conditions in metastatic cancer cells and its implication in EMT [J].Eur J Cell Biol,2017,96(2):164-171.
[14] 季律,魏盼,王璨,等.麻黄-甘草药对对脂多糖诱导的RAW264.7细胞的体外抗炎作用分析[J].中国实验方剂学杂志,2017,23(18):83-88.
[15] 魏盼,季律,袁为远,等.麻黄-甘草药对对小鼠脾细胞的增殖以及Th1/Th2平衡的影响[J].南京中医药大学学报,2018,34(4):371-375,380.