机械牵张对人肺上皮细胞纤维增殖的影响

Effects of mechanical stretch on fiber proliferation in human lung epithelial cells

背景:机械通气是目前重症呼吸疾病重要有效的治疗支持手段之一,但不适当的机械通气可使肺损伤加重,甚至纤维化形成。如何最大程度减少呼吸机相关肺损伤,发挥呼吸机的真正保护作用显得至关重要。肺是一个力学器官,机械通气使肺泡反复充气,对邻近的肺上皮细胞产生牵张作用,使其处于一个非正常的损伤修复过程。因此,过度机械牵张在促进肺纤维增殖发生中可能扮演重要角色。目前国内外关于机械牵张对肺上皮细胞纤维增殖的影响报道较少,其具体发生机制并不清楚。目的:观察不同机械牵张强度及牵张时间对人肺上皮BEAS-2B细胞中转化生长因子β1、波形蛋白及Ⅰ型胶原表达的影响。方法:体外培养人肺上皮细胞株BEAS-2B,取对数生长期的细胞分为静止对照组、10%牵张组、20%牵张组。采用FX-5000T细胞应力加载系统对人肺上皮BEAS-2B细胞以频率20次/min,正弦波形式分别牵张24,48,72 h,采用倒置显微镜观察细胞形态变化,RT-qPCR和免疫荧光方法检测转化生长因子β1、Ⅰ型胶原mRNA表达及波形蛋白表达变化。结果与结论:①静止对照组BEAS-2B细胞呈卵圆形贴壁生长,20%牵张组牵张72 h后细胞形态由鹅卵圆形变为长梭样,细胞间隙增宽;②随着机械牵张强度的增加,BEAS-2B细胞中转化生长因子β1、Ⅰ型胶原mRNA表达及波形蛋白水平较静止对照组均增加,并呈时间依赖趋势(P<0.05);与静止对照组比较,10%牵张组转化生长因子β1、Ⅰ型胶原mRNA及波形蛋白变化不明显;③结果表明,过度机械牵张可刺激人肺上皮BEAS-2B细胞中转化生长因子β1、波形蛋白及Ⅰ型胶原表达增加,过度机械牵张在促进肺纤维增殖发生中发挥重要作用。

BACKGROUND:Mechanical ventilation is currently one of important and effective treatment and support methods for severe respiratory diseases,but improper mechanical ventilation can aggravate lung injury and even form fibrosis.How to minimize the lung damage associated with the ventilator and play real protective role of the ventilator is very important.The lung is a mechanical organ.Mechanical ventilation causes the alveoli to be inflated repeatedly,which stretches the neighboring lung epithelial cells,and puts it in an abnormal damage repair.Therefore,excessive mechanical stretch may play an important role in promoting the occurrence of lung fibrosis.At present,there are few reports on the effect of mechanical stretch on the proliferation of lung epithelial cells at home and abroad,and the specific mechanism is not clear.OBJECTIVE:To investigate the effect of different mechanical stretch strengths and stretch time on expression of transforming growth factorβ1,vimentin,and type I collagen in human lung epithelial BEAS-2B cells.METHODS:Human lung epithelial BEAS-2B cells were cultured in vitro.Cells at logarithmic growth phase were divided into control static group,10%stretch group,and 20%stretch group.The FX-5000 system was used to stretch BEAS-2B cells at 20 times/min and sine wave for 24,48,and 72 hours.The morphological changes in cells were observed with the inverted microscope.The expression levels of transforming growth factorβ1,vimentin,and typeⅠcollagen mRNA were evaluated by RT-qPCR and immunofluorescence.RESULTS AND CONCLUSION:(1)BEAS-2B cells displayed cobblestone morphology and linked closely in the control static group.The cell morphology changed from cobblestone shape into long spindle,and intercellular space increased obviously after 20%mechanical stretch for 72 hours.(2)The expression of mRNA and protein of transforming growth factorβ1,vimentin,and typeⅠcollagen in human lung epithelial BEAS-2B cells was up-regulated with increasing stretch force in a time-dependent manner compared with the control static group(P<0.05).Compared with the control static group,the changes in transforming growth factorβ1,vimentin,and typeⅠcollagen were not significant in the 10%stretch group.(3)It is concluded that mechanical stretch increases the expression of transforming growth factorβ1,vimentin,and typeⅠcollagen in human lung epithelial BEAS-2B cells.Excessive mechanical stretch plays an important role in promoting lung fiber proliferation.