机械牵张对人肺上皮细胞转分化的影响

Effects of cyclic stretch on the induction of the transdifferentiation in human lung epithelial cells

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.

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.