CAJ | 학술논문

目的研究周期性机械牵张刺激对人肺上皮细胞BEAS-2B向间质转分化的影响.方法应用FX-5000T细胞牵张加载系统对BEAS-2B细胞以0.33 Hz频率分别施加10％或20％牵张应力24、48和72 h,相差显微镜下观察细胞形态变化；应用免疫荧光双标染色和实时荧光定量聚合酶链反应(qRT-PCR)分别检测牵张前后E-钙黏蛋白(E-cadherin)、细胞角蛋白-8(CK-8)、α-平滑肌肌动蛋白(α-SMA)和波形蛋白(Vimentin)的蛋白及mRNA表达情况.结果 ①20％病理牵张48 h后,BEAS-2B细胞开始由鹅卵圆形变为长梭形,牵张72 h细胞间隙明显增宽,细胞长梭形改变更明显.10％生理牵张下细胞形态无明显改变.②免疫荧光双标染色结果显示,20％牵张72 h时CK-8蛋白表达明显下调,E-cadherin表达明显下调,α-SMA蛋白表达明显增强.③与未牵张对照组(作为1)相比,20％牵张组E-cadherin mRNA表达在牵张48 h和72 h分别下调至0.388±0.056和0.247±0.064(均P＜0.05),CK-8 mRNA表达在牵张72 h下调至0.436 ±0.060 (P＜0.01),α-SMA mRNA表达在牵张48 h和72 h分别上调至1.437±0.267和1.261±0.247(均P＜0.05),VimentinmRNA表达在牵张48 h上调至1.679±0.172(P＜0.05).10％牵张组E-cadherin mRNA表达在牵张72 h下调至0.387±0.081(P＜0.05),Vimentin mRNA表达在牵张48 h上调至1.688±0.179(P＜0.05)；其余各指标变化不明显,差异无统计学意义.结论持续过度牵张能诱导人肺上皮细胞BEAS-2B发生上皮向间质转分化.
목적 연구주기성궤계견장자격대인폐상피세포BEAS-2B향간질전분화적영향.방법 응용FX-5000T세포견장가재계통대BEAS-2B세포이0.33 Hz빈솔분별시가10％혹20％견장응력24、48화72 h,상차현미경하관찰세포형태변화；응용면역형광쌍표염색화실시형광정량취합매련반응(qRT-PCR)분별검측견장전후E-개점단백(E-cadherin)、세포각단백-8(CK-8)、α-평활기기동단백(α-SMA)화파형단백(Vimentin)적단백급mRNA표체정황.결과 ①20％병리견장48 h후,BEAS-2B세포개시유아란원형변위장사형,견장72 h세포간극명현증관,세포장사형개변경명현.10％생리견장하세포형태무명현개변.②면역형광쌍표염색결과현시,20％견장72 h시CK-8단백표체명현하조,E-cadherin표체명현하조,α-SMA단백표체명현증강.③여미견장대조조(작위1)상비,20％견장조E-cadherin mRNA표체재견장48 h화72 h분별하조지0.388±0.056화0.247±0.064(균P＜0.05),CK-8 mRNA표체재견장72 h하조지0.436 ±0.060 (P＜0.01),α-SMA mRNA표체재견장48 h화72 h분별상조지1.437±0.267화1.261±0.247(균P＜0.05),VimentinmRNA표체재견장48 h상조지1.679±0.172(P＜0.05).10％견장조E-cadherin mRNA표체재견장72 h하조지0.387±0.081(P＜0.05),Vimentin mRNA표체재견장48 h상조지1.688±0.179(P＜0.05)；기여각지표변화불명현,차이무통계학의의.결론 지속과도견장능유도인폐상피세포BEAS-2B발생상피향간질전분화.
Objective To investigate the effect of mechanical stretch induced epithelial-mesenchymal transition in human lung epithelial cells BEAS-2B in vitro.Methods The human lung epithelial cells BEAS-2B were subjected to cyclic stretch by the FX-5000T system at 0.33 Hz of 10％ or 20％ elongation for 24,48 and 72 hours respectively.The morphologic changes were observed by microscopy.The mRNA and protein expressions of E-cadherin,Cytokeratin-8 (CK-8),α-smooth muscle actin (α-SMA) and Vimentin were evaluated by immunofluorescence before and after mechanical stretch and fluorescent quantitation reverse transcription-polymerase chain reaction (qRT-PCR).Results ① When stretch by 20％ elongation for 48 hours,the morphological changes in BEAS-2B cells from cobblestone-like structure to elongated shape and obviously when stretch for up to 72 hours,while 10％ elongation showed no significant morphological changes comparing to control.② Decreasing E-cadherin and CK-8 protein expression was associated with increased immunostaining for α-SMA protein at 72 hours after 20％ mechanical stretch.③ Expression of E-cadherin mRNA was decreased to 0.388 ± 0.056 and 0.247 ± 0.064 after 20％ mechanical stretch for 48 hours and 72 hours compared with control without stretch (set 1,both P＜0.05),expression of CK-8 mRNA was decreased to 0.436 ± 0.060 at 72 hours after 20％ mechanical stretch (P＜0.01),α-SMA mRNA was increased to 1.437 ± 0.267 (48 hours) and 1.261 ± 0.247 (72 hours) after 20％ mechanical stretch (both P＜0.05),and Vimentin mRNA was increased to 1.679 ± 0.172 (48 hours) after 20％ mechanical stretch (P＜ 0.05).Expression of E-cadherin mRNA was decreased to 0.387 ± 0.081 at 72 hours after 10％ mechanical stretch (P＜0.05),Vimentin mRNA was increased to 1.688 ± 0.179 at 48 hours after 10％ mechanical stretch while other markers showed no significant changes comparing with control.Conclusion Excessive mechanical stretch could induce epithelial-mesenchymal transition in lung epithelial cells BEAS-2B in vitro.