高氧暴露对新生鼠Ⅱ型肺泡上皮细胞转分化水平的调节作用

The regulatory effect of hyperoxia exposure on the transdifferentiation level of type II alveolar epithelial cells in newborn rats

目的研究体内及体外高氧暴露对Ⅱ型肺泡上皮细胞（typeⅡ alveolar epithelial cells，AECⅡ）转分化水平的影响，旨在阐明支气管肺发育不良（bronchopulmonary dysplasia，BPD）肺上皮损伤的发生机制。方法新生Wistar大鼠生后随机分为对照组（吸入空气）和模型组（吸入氧浓度为85％），于7d、14d、21d进行动物模型肺组织取材并分离AECⅡ。细胞标本检测Ⅰ型肺泡上皮细胞（type Ⅰalveolar epithelial cells，AECI）特异性标志物水通道蛋白5（aquaporin5，AQP5）及AECⅡ标志物表面活性蛋白C（surfactant proteinC，SP-C）表达。从正常新生鼠肺分离的AECⅡ在体外原代培养24h后随机分为常氧组（21％O2）和高氧组（85％O2），培养48h后收集细胞，应用免疫荧光双标染色观察AQP5和SP-C表达及定位，Western blot方法检测AQP5和SP—C蛋白表达水平，荧光实时定量PCR方法检测AQP5和SP-C mRNA表达水平。结果BPD模型组大鼠AECⅡ中AQP5蛋白表达从7d开始较对照组增多，SP-C蛋白表达从14d开始较对照组减少。模型组中AQP5mRNA从7d开始表达增多，SP-C mRNA从7d开始表达减少（P〈0．05），且随高氧暴露时间延长两组间差异更加显著。由正常新生鼠肺分离的AECII进行体外原代培养后，免疫荧光双染可见高氧组较常氧组AQP5表达增多，SP-C表达减少，双染细胞明显增多。蛋白和mRNA定量检测结果均提示高氧组较常氧组AQP5表达增多，SP-C表达减少（P〈0．01）。结论无论体内还是体外高氧暴露下，AECⅡ特异性标志物SP—C表达下调，而AECⅠ特异性标志物AQP-5表达上调，提示AECⅡ过度转分化参与高氧肺损伤后的修复过程。

Objective To investigate the effect of hyperoxia on the transdifferentiation level of type Ⅱ alveolar epithelial cells （ AEC Ⅱ ） in vivo and in vitro, in order to illuminate the mechanism of epithelial injury in bronchopulmonary dysplasia （BPD）. Methods Newborn Wistar rats were randomly devided into control group （room air inhalation） or model group （85% oxygen inhalation） after birth. Lung tissue sampling and AEC Ⅱ isolation was conducted on 7 d, 14 d, 21 d. Type Ⅰ alveolar epithelial cells （ AEC Ⅰ ） marker aquaporin 5 （ AQP5 ） and AEC H marker surfactant protein C （SP-C） were examined by Western blot and florescent real-time PCR. AEC Ⅱ isolated from normal newborn rats was randomly devided into normoxia group （21% oxygen） or hyperoxia group （85% oxygen） after 24 h culture, and continued culturing for another 48 h in vitro. Then the morphological changes of cells were observed under inverted phase contrast microscope. The expression and location of markers for AEC Ⅰ and AEC Ⅱ was examined by double staining. The protein expression of AQP5 and SP-C was evaluated by Western blot, and the mRNA expression of these markers was examined by florescent real-time PCR. Results In AEC II isolated from the an- imal models, the AQP5 protein expression increased from 7 d while the SP-C expression decreased from 14 d in the model group comparing with the control group. In the model group, AQP5 mRNA expression increased and SP-C mRNA expression decreased since 7 d after hyperoxia exposure （ P 〈 0. 05 ）, with the difference between groups more obvious as exposure time extending. After culturing in vitro, AEC Ⅱ isolated from normal new- born rats expressed more AQP5 and less SP-C, with more cells double stained in the hyperoxia group compared with the normoxia group, examined by immunofluorescence double staining. The protein and mRNA examination results both showed that AQP5 expression increased and SP-C expression decreased in the hyperoxia group compared with the normoxia group （P 〈 0. 01 ）. Condusion After hyperoxia exposure, no matter in vivo or in vitro, the expression of AEC Ⅱ marker SP-C decreases while the expression of AEC Ⅰ marker AQP5 increases. These results indicate that the excessive transdifferentiation of AEC Ⅱ takes part in the recovery process after hyperoxia induced lung injury.