目的 探讨上皮-间充质转化（epithelial-mesenchymal transition, EMT）在大鼠支气管肺发育不良（bronchopulmonary dysplasia, BPD）模型中的作用及机制。 方法 实验分为两部分。（1）将48只早产大鼠随机分为常氧组和高氧组，每组24只。高氧组暴露于85％氧气中建立早产大鼠BPD模型，常氧组置于同一室内常压空气中，于实验第1、4、7、14天收集早产大鼠肺组织标本。（2）将大鼠肺泡Ⅱ型上皮细胞系（RLE-6TN）随机分为常氧组（常规空气中培养）和高氧组（在95%氧气中培养），分别于高氧暴露后12 h、24 h、48 h收集细胞标本进行检测。使用苏木精-伊红染色法观察早产大鼠肺泡化情况，免疫荧光检测早产大鼠肺组织和RLE-6TN细胞肺表面活性蛋白C（surfactant protein C, SPC）和α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）的共定位。实时荧光定量聚合酶链反应和蛋白免疫印迹法检测早产大鼠肺组织和RLE-6TN细胞EMT相关mRNA和蛋白表达水平。 结果 （1）与常氧组相比，高氧暴露7 d开始高氧组早产鼠可见肺泡化阻滞和肺泡结构简单化改变；肺组织中SPC和α-SMA存在明显共定位现象，高氧暴露7 d、14 d时，与常氧组比较，高氧组SPC表达减少，α-SMA表达增加；高氧暴露7 d、14 d时，与常氧组比较，高氧组TGF-β1、α-SMA、N-钙黏蛋白mRNA和蛋白表达水平升高（P<0.05），SPC、E-钙黏蛋白mRNA和蛋白表达水平降低（P<0.05）。（2）高氧暴露24 h、48 h时，与常氧组比较，高氧组SPC表达减少，α-SMA表达增加；高氧暴露48 h时，与常氧组比较，高氧组SPC、E-钙黏蛋白mRNA和蛋白表达降低（P<0.05），TGF-β1、α-SMA、N-钙黏蛋白mRNA和蛋白表达升高（P<0.05）。 结论 EMT破坏了BPD早产大鼠肺泡上皮细胞之间的紧密连接，造成肺泡结构简单化和发育异常，参与了BPD的发生发展。
Abstract
Objective To investigate the role and mechanism of epithelial-mesenchymal transition (EMT) in a rat model of bronchopulmonary dysplasia (BPD). Methods The experiment consisted of two parts. (1) Forty-eight preterm rats were randomly divided into a normoxia group and a hyperoxia group, with 24 rats in each group. The hyperoxia group was exposed to 85% oxygen to establish a BPD model, while the normoxia group was kept in room air at normal pressure. Lung tissue samples were collected on days 1, 4, 7, and 14 of the experiment. (2) Rat type II alveolar epithelial cells (RLE-6TN) were randomly divided into a normoxia group (cultured in air) and a hyperoxia group (cultured in 95% oxygen), and cell samples were collected 12, 24, and 48 hours after hyperoxia exposure. Hematoxylin-eosin staining was used to observe alveolarization in preterm rat lungs, and immunofluorescence was used to detect the co-localization of surfactant protein C (SPC) and α-smooth muscle actin (α-SMA) in preterm rat lung tissue and RLE-6TN cells. Quantitative real-time polymerase chain reaction and protein immunoblotting were used to detect the expression levels of EMT-related mRNA and proteins in preterm rat lung tissue and RLE-6TN cells. Results (1) Compared with the normoxia group, the hyperoxia group showed blocked alveolarization and simplified alveolar structure after 7 days of hyperoxia exposure. Co-localization of SPC and α-SMA was observed in lung tissue, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 7 and 14 days of hyperoxia exposure compared to the normoxia group. In the hyperoxia group, the mRNA and protein levels of TGF-β1, α-SMA, and N-cadherin were increased, while the mRNA and protein levels of SPC and E-cadherin were decreased at 7 and 14 days of hyperoxia exposure compared to the normoxia group (P<0.05). (2) SPC and α-SMA was observed in RLE-6TN cells, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 24 and 48 hours of hyperoxia exposure compared to the normoxia group. Compared to the normoxia group, the mRNA and protein levels of SPC and E-cadherin in the hyperoxia group were decreased, while the mRNA and protein levels of TGF-β1, α-SMA, and E-cadherin in the hyperoxia group increased at 48 hours of hyperoxia exposure (P<0.05). Conclusions EMT disrupts the tight connections between alveolar epithelial cells in a preterm rat model of BPD, leading to simplified alveolar structure and abnormal development, and is involved in the development of BPD. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 765-773
关键词
支气管肺发育不良 /
上皮-间充质转化 /
肺泡Ⅱ型上皮细胞 /
紧密连接 /
早产大鼠
Key words
Bronchopulmonary dysplasia /
Epithelial-mesenchymal transition /
Type Ⅱ alveolar epithelial cell /
Tight junction /
Preterm rat
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参考文献
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基金
上海市“科技创新行动计划”医学创新研究专项基金。
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