Notch1/Hes1信号调控CCAAT/增强子结合蛋白α表达对高氧损伤肺泡Ⅱ型上皮细胞增殖与分化的影响

Effect of Notch1/Hes1 signal regulating the expression of CCAAT/enhancer-binding protein alpha on the proliferation and differentiation of type II alveolar epithelial cells induced by hyperoxia

背景:Notch1/Hes1信号和CCAAT/增强子结合蛋白α(CCAAT/enhancer-binding protein alpha,C/EBPα)在肺发育及肺损伤中均发挥重要作用。前期研究发现,高氧可引起肺泡Ⅱ型上皮细胞(typeⅡalveolar epithelialcell,AECII)增殖分化异常,并与C/EBPα异常表达有关,而这种异常是否与Notch1/Hes1信号调控有关,目前尚不清楚。目的:探讨Notch1/Hes1信号调控C/EBPα表达对高氧损伤AECII增殖与分化的影响。方法:体外培养人AECII,将细胞随机分为空气组、空气激活剂组(Notch通路激活剂Jagged1蛋白500μg/L)、高氧组(按3 L/min通入体积分数95%O_2和5%CO_2高纯混合气体且持续10 min)、高氧激活剂组(在通氧前30min加入Notch通路激活剂Jagged1蛋白500μg/L)。于干预后24h收获各组细胞,采用RT-PCR和Western blot法分别检测Notch1、Hes1及C/EBPαmRNA与蛋白表达水平;CCK-8法检测AECII细胞增殖;流式细胞术标记肺泡Ⅰ型上皮细胞特异性表面标志蛋白AQP5检测AECII分化。结果与结论:与空气组比,空气激活剂组Notch1、Hes1、C/EBPαmRNA和蛋白表达显著增加(P <0.05),AECII增殖增加而分化减少(P<0.05);高氧组和高氧激活剂组Notch1、Hes1、C/EBPαmRNA和蛋白表达显著降低(P <0.05),AECII增殖减少而分化增加(P <0.05);与高氧组比,高氧激活剂组Notch1、Hes1、C/EBPαmRNA和蛋白表达增加(P <0.05),AECII增殖增加而分化减少(P <0.05);结果提示,Notch1/Hes1信号可调控C/EBPα表达并能影响高氧损伤AECII增殖与分化功能。

BACKGROUND:Notch1/Hes1 signal and CCAAT/enhancer-binding protein alpha(C/EBPα)play important roles in lung development and injury.Preliminary studies have shown that hyperoxia can induce abnormal proliferation and differentiation of type II alveolar epithelial cells(AECII),which is related to C/EBPαexpression.But,whether Notch1/Hes1 signal is involved remains unclear.OBJECTIVE:To investigate the effect of Notch1/Hes1 signal regulating C/EBPαon the proliferation and differentiation of AECII induced by hyperoxia.METHODS:Human AECIIs were cultured in vitro,and randomly divided into air group,air activator group(Notch pathway activator Jagged1 protein,500μg/L),hyperoxia group(95%O2 and 5%CO2 mixture gas administered at the speed of 3 L/minute for 10 minutes),hyperoxia activator group(500μg/L Notch pathway activator Jagged1 protein added at 30 minutes before oxygen administration).AECIIs of each group were collected after 24 hours of intervention.The expression of Notch1,Hes1 and C/EBPαat mRNA and protein levels was determined by RT-PCR and western blot assay.The proliferation of AECII was measured by cell counting kit-8 assay.Flow cytometry labeled AQP5 was applied to detect the differentiation of AECII.RESULTS AND CONCLUSION:Compared with the air group,the mRNA and protein expression levels of Notch1,Hes1 and C/EBPαwere increased significantly in the air activator group(P<0.05),the proliferation of AECII was increased and the differentiation was reduced significantly(P<0.05).In the hyperoxia and hyperoxia activator groups,the mRNA and protein expression levels of Notch1,Hes1 and C/EBPαwere significantly decreased,the proliferation of AECII was decreased and the differentiation was increased significantly(P<0.05).Compared with the hyperoxia group,the mRNA and protein expression levels of Notch1,Hes1 and C/EBPαwere significantly increased in the hyperoxia activator group(P<0.05),and the proliferation of AECII was increased and the differentiation was decreased significantly(P<0.05).These results suggest that Notch1/Hes1 signal regulates the expression of C/EBPαand affects the proliferation and differentiation of AECII after exposure to hyperoxia.