摘要
目的探讨核转录因子Snail介导的肺上皮间质转化(epithelial-mesenchymal transition,EMT)在肌成纤维细胞活化中的作用。方法培养小鼠Ⅱ型肺泡上皮细胞(MLE-12)并利用转化生长因子-β1(transforming growth factor-β1,TGF-β1)刺激获得EMT模型,应用RT-qPCR检测上皮标志物E钙粘蛋白(E-cadherin,CDH1)、间质标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、核转录因子Snail mRNA表达变化。建立MLE-12与小鼠胚肺成纤维细胞(NIH-3T3)共培养体系,应用RT-qPCR检测NIH-3T3的α-SMA、Ⅰ型胶原(collagenⅠα1,COL1A1)、Ⅲ型胶原(collagenⅢα1,COL3A1)mRNA表达。利用Snail-shRNA转染MLE-12,应用RT-qPCR、Western blotting检测Snail mRNA和蛋白质表达。建立慢病毒转染的MLE-12与NIH-3T3共培养体系,应用RT-qPCR检测NIH-3T3的α-SMA、COL1A1、COL3A1 mRNA表达。采用单因素方差分析及最小显著差法判断结果差异是否具有统计学意义。结果加入TGF-β1刺激48 h后,RT-qPCR结果显示,与对照组相比,TGF-β1组的CDH1 mRNA的表达下调、α-SMA和Snail mRNA的表达上调(P<0.05);共培养体系中,RT-qPCR结果显示,与TGF-β1和MLE-12组相比,加入TGF-β1刺激的MLE-12引起下室NIH-3T3的α-SMA、COL1A1、COL3A1 mRNA表达上调,且上调程度高于TGF-β1和MLE-12的单独作用(P<0.05);慢病毒转染后,与阴性对照病毒组相比,Snail慢病毒干扰组Snail的mRNA和蛋白质表达均明显下调(P<0.05);分别用阴性对照病毒和Snail慢病毒转染MLE-12后与NIH-3T3建立共培养体系,RT-qPCR结果显示,与TGF-β1+阴性对照病毒组相比,TGF-β1+Snail慢病毒转染组下室NIH-3T3的α-SMA、COL1A1、COL3A1 mRNA表达上调程度降低(P<0.05)。结论Snail介导的小鼠Ⅱ型肺泡上皮细胞发生EMT可引起肌成纤维细胞活化,敲减Ⅱ型肺泡上皮细胞的Snail基因可抑制成纤维细胞活化为肌成纤维细胞。提示Snail介导的肺上皮间质转化在肌成纤维细胞活化中发挥重要作用。
Objective To explore the effect of nuclear transcription factor Snail-mediated lung epithelial-mesenchymal transition(EMT)in activation of myofibroblasts.Methods MLE-12 cells(murine epithelial cell line)were stimulated by transforming growth factor-β1(TGF-β1)to build EMT model,the mRNA expression of epithelial marker CDH1,mesenchymal marker vimentin and nuclear transcription factor Snail were detected by RT-qPCR.MLE-12 cells were co-cultured with mouse embryo fibroblast(NIH-3T3)cells.The mRNA expression ofα-smooth muscle actin(α-SMA),collagenⅠα1(COL1A1),collagenⅢα1(COL3A1)of NIH-3T3 were detected by RT-qPCR.MLE-12 cells were transfected with Snail-shRNA lentivirus.The expression of Snail mRNA and protein of MLE-12 cells were detected by RT-qPCR and Western blotting.The Snail-shRNA-transfected-MLE-12 cells were co-cultured with NIH-3T3 cells.The mRNA expression ofα-SMA,COL1A1,COL3A1 of NIH-3T3 were detected by RT-qPCR.Multiple groups were analyzed by one-way ANOVA and LSD test to determine significant differences between groups at P<0.05.Results The results of RT-qPCR showed that after stimulated by TGF-β1 for 48 hours,compared with control,the mRNA expression of CDH1 of MLE-12 in TGF-β1 group was down-regulated whileα-SMA and Snail were up-regulated(P<0.05).In the co-culture model,the results of RT-qPCR showed that compared to TGF-β1 and MLE-12 group,MLE-12 stimulated by TGF-β1 cause up-regulation ofα-SMA,COL1A1,COL3A1 mRNA expression of NIH-3T3(P<0.05).After transfected with Snail-shRNA lentivirus,mRNA and protein expression of MLE-12 were down-regulated compared with control virus group(P<0.05).In the co-culture model of lentivirus-transfected-MLE-12 and NIH-3T3,results of RT-qPCR showed thatα-SMA,COL1A1,COL3A1 mRNA up-regulation of NIH-3T3 in TGF-β1+Snail-shRNA group were lower than TGF-β1+control-shRNA group(P<0.05).Conclusion Snail-mediated EMT of MLE-12 can cause activation of myofibroblasts,knockdown of Snail in MLE-12 can inhibit fibroblasts activation into myofibroblasts,suggested Snail-mediated lung epithelial-mesenchymal transition plays an important role in myofibroblasts activation.
作者
李思泠
朱钟慧
李秋月
许春杰
赵静
王炎
田琳
Li Siling;Zhu Zhonghui;Li Qiuyue;Xu Chunjie;Zhao Jing;Wang Yan;Tian Lin(Department of Occupational Health and Environmental Health,School of Public Health,Capital Medical University,Beijing 100069,China)
出处
《首都医科大学学报》
CAS
北大核心
2020年第1期92-98,共7页
Journal of Capital Medical University
基金
国家自然科学基金(81703197,81273047,91643114,81472958,81602832)~~
