新生鼠高氧性肺损伤模型中肺泡上皮细胞表达密闭小带蛋白-1及密闭小带蛋白-1相关核酸结合蛋白的研究

Study on the expression level of zonula occludens 1 and zonula occludens 1 related nucleic acid binding protein in alveolar epithelial cells in hyperoxia induced bronchopulmonary dysplasia newborn rats

目的 以往研究已发现高氧性肺损伤时肺泡上皮细胞(alveolar epithelial cell,AECs)存在增殖及转分化异常,本研究旨在明确高氧性肺损伤模型中AECs表达密闭小带蛋白-1( zonula occludens 1,ZO-1)、ZO-1相关核酸结合蛋白(ZO-1-related nucleic acid binding proteins,ZONAB)的情况,探讨其对肺损伤时肺组织中AECs增殖及转分化的影响.方法 足月新生Wistar大鼠于生后12 h内随机分为2组:模型组(吸入氧浓度为85%)和对照组(吸入空气).于暴露7、14、21 d留取肺组织标本,采用组织免疫荧光双标染色观察Ⅱ型肺泡上皮细胞( type Ⅱ alveolar epithelial cells,AECⅡ)表达ZONAB情况.同时,于各时间点分离动物模型肺组织中AECⅡ,通过Western blot和Real-Time PCR等方法检测两组肺组织中及AECⅡ中ZO-1、ZONAB蛋白和mRNA表达水平.另外,从正常新生鼠肺组织分离AECⅡ,分为模型组(氧浓度85%)和对照组(氧浓度21%),在体外培养48 h后检测ZO-1、ZONAB蛋白和基因表达水平,并进行细胞免疫荧光染色观察ZONAB的表达量及表达部位.结果 肺组织免疫荧光双标染色可见模型组AECⅡ表达ZONAB较对照组显著减少.从动物模型肺组织分离的AECⅡ标本中, ZO-1、ZONAB的蛋白及mRNA表达水平从高氧暴露7 d开始较对照组明显减少.从正常新生鼠肺组织分离AECⅡ,在体外高氧条件下培养48 h后,ZO-1、ZONAB的蛋白及mRNA表达水平均较对照组显著降低.细胞免疫荧光染色结果发现,对照组中ZONAB表达较多,且表达部位多位于细胞连接处及细胞核内,而模型组ZONAB表达量较对照组明显减少,且表达部位聚集于胞浆,细胞连接处及核内均表达很少.结论 ZO-1作为紧密连接相关蛋白,其在高氧性肺损伤模型中表达下调,除了破坏肺上皮屏障介导肺水肿发生外,还通过调节转录因子ZONAB表达从而参与AECs增殖及分化的调节,提示这可能是导致高氧性肺损伤发生的又一途径.

Objective Previous studies have found abnormal proliferation and transdifferentiation of alveolar epithelial cells(AECs)in hyperoxic lung injury of neonatal rats.The purpose of this study was to clarify the expression of zonula occludens 1(ZO-1)and ZO-1 related nucleic acid binding protein(ZONAB)in AECs in hyperoxic lung injury model,in order to investigate its effect on the proliferation and transdifferentiation of AECs in the injured lung tissue.Methods Full-term neonatal Wistar rats were randomly divided into two groups within 12 h after birth,model group(inhaled oxygen concentration 85%)and control group(inhaled air).Lung specimens were collected at 7,14 and 21 days after exposure.The expression of ZONAB in typeⅡalveolar epithelial cells(AECⅡ)was observed by double immunofluorescence staining.At the same time,AECⅡwas isolated from lung tissues of animal models at these time points,and the expression levels of ZO-1,ZONAB protein and mRNA in lung tissues and AECⅡof the two groups were detected by Western blot and Real-Time PCR.In addition,AECⅡwas isolated from lung tissue of normal newborn rats and then divided into model group(85%oxygen concentration)and control group(21%oxygen concentration).After 48 hours of culture in vitro,the expression levels of ZO-1,ZONAB protein and mRNA were detected,and the expression level and location of ZONAB were observed by immunofluorescence staining.Results Double immunofluorescence staining showed that the expression of ZONAB in AECⅡin model group was significantly lower than that in control group.The protein and mRNA expression levels of ZO-1 and ZONAB in AECⅡisolated from lung tissue of model group were both significantly lower than those from control group,starting from 7 d after hyperoxia exposure.AECⅡisolated from lung tissue of normal newborn rats,were then incubated for 48 hours under hyperoxia or normoxia in vitro,the protein and mRNA expression levels of ZO-1 and ZONAB significantly decreased in model group compared with those in control group.The results of immunofluorescence staining showed that the expression of ZONAB was higher in AECⅡof the control group,and ZONAB was mostly located in the junction and nucleus of cells,while the expression of ZONAB in the model group significantly decreased than that in the control group,and the expression sites were clustered in the cytoplasm,with little expression in the junction and nucleus.Conclusion ZO-1,as a tight junction-related protein,is down-regulated in hyperoxic lung injury model.In addition to destroying pulmonary epithelial barrier to mediate pulmonary edema,it also participated in the regulation of proliferation and differentiation of AECs by regulating transcription factor ZONAB,suggesting that this may be another pathway leading to hyperoxic lung injury.