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

Study on Establishment of Epithelial-Mesenchymal Transition and Regulation of Mahuang-Gancao Couplet Medicines in 16HBE Cells

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ECadherin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NCadherin、α-SMA的表达,并恢复ECadherin蛋白的表达,对ZO1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 16HBE cells and to investigate the effects of couplet medicines on EMT in 16HBE cells. METHODS Western blot was applied to detect the mark proteins expression in 16HBE 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 16HBE 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 16HBE 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 16HBE 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 16HBE cells.