目的 探讨转录因子CASZ1在高氧致支气管肺发育不良新生大鼠肺组织中的表达及其对肺微血管发育的关系.方法 足月新生Sprague Dawley (SD)大鼠48只,雌雄不限,在出生24h内按随机数字法分为实验组和对照组,每组24只.实验组暴露于高氧[吸入氧浓度(FiO2):0.80±0.02]氧箱中,对照组置于空气中(FiO2:0.21).两组分别于实验开始后3、7和14 d随机数字法选取8只处死并取出肺组织.HE染色光镜下观察其肺组织形态结构改变,采用放射状肺泡计数(RAC)和呼吸膜厚度来评价肺泡发育情况;免疫组织化学方法检测CD31的表达并计算肺微血管密度;免疫组织化学、Western印迹、实时荧光定量PCR检测肺组织CASZ1定位、分布及表达.结果 HE染色显示:实验组大鼠随着日龄的增加肺泡出现融合断裂、呼吸膜增厚、RAC明显少于对照组[14 d:呼吸膜厚度(12.69 ±0.63)/μm比(6.53±0.16) μm,RAC(5.9±0.4)个比(8.4±1.0)个,t=19.046、4.760,P均=0.000].免疫组织化学显示:CD31蛋白主要表达于肺微血管内皮细胞中,实验组3、7、14 dCD31阳性细胞的平均积分光密度值均明显低于对照组[(16.6±1.6)×103比(40.1 ±2.4)×103、(18.1±1.4)×103比(83.2±5.2)×103, (49.2±5.4)× 103比(136.2±28.1)×103,t=16.185、16.066、6.078,P均＜0.01];肺微血管密度也显著低于对照组[(3.84±0.15)％比(6.01±0.22)％,(4.17±0.38)％比(6.15±0.24)％, (5.43±0.44)％比(9.13±0.25)％,t=16.124、8.773、14.076,P均＜0.01].实时荧光定量PCR及Western印迹结果显示:各时间点实验组新生大鼠肺组织CASZ1mRNA水平均显著低于对照组(0.56 ±0.17比1.00±0.26、0.32±0.29比0.58 ±0.14、0.4±0.22比0.56±0.15,t=3.890、3.303、2.388,P均＜0.05);CASZ1蛋白表达水平也均明显低于对照组(0.65±0.02比0.78±0.23、0.46±0.03比0.75 ±0.05、0.34±0.22比0.75 ±0.04,t =6.200、10.485、14.998,P均＜0.05).实验组CASZ1蛋白表达水平与肺微血管密度成显著正相关(r=0.519、P＜0.01).结论 CASZI参与了新生大鼠支气管肺发育不良发生发展,且与其肺微血管发育障碍密切相关.%Objective To explore the expression of CASZ1 and its relationship with the pulmonary microvascular development in lung tissue of newborn rats exposed to hyperoxia which induced bronchopulmonary dysplasia (BPD).Method Forty-eight newborn Sprague Dawley(SD) rats (male and female unlimited) were randomly divided into two groups:experimental group and control group according to random digits table with 24 in each.The rats in experimental group were exposed to high oxygen volume fraction of 800 ml/L and the rats in control group were exposed to normal air.Eight rats were randomly selected from each group on day 3 and 7 after oxygen exposure.The sections of lung were stained with HE method in order to assess lung histological changes, the alveolar development was evaluated by the number of radial alveolar count (RAC) and septal wall thickness.CD31 was detected by immunohistochemistry (IHC) method and the capillary density was calculated.The location, distribution and expression of CASZ1 in the lung tissue were detected by the immunohistochemistry, Western blotting, and quantitative PCR (qPCR).Result (1) Stained by HE, lungs of experimental group showed destroyed alveoli, alveoli fusion and increased septal wall thickness, RAC were significantly lower than those in control group(14 d:septal wall thickness (12.69±0.63) μmvs.(6.53±0.16) μm,RAC 5.9 ±0.4 vs.8.4±1.0,t=19.046,4.760, P both =0.000).(2) CD31 protein was expressed predominantly in cytoplasm of pulmonary microvascular endothelial cells.The experimental group CD31 average optical density (AIOD) were decreased compared with control group((16.6 ± 1.6) ×103 vs.(40.1 ±2.4) × 103,(18.1 ±1.4) ×103 vs.(83.2 ±5.2) × 103,(49.2 ±5.4) × 103 vs.(136.2 ± 28.1) × 103,t =16.185, 16.066 and 6.078,P ＜ 0.01 for all comparisons).Capillary density in experimental group was also significantly decreased compared with control group ((3.84 ±0.15)％ vs.(6.01 ±0.22)％, (4.17 ±0.38)％ vs.(6.15 ±0.24)％, (5.43 ±0.44)％ vs.(9.13 ±0.25)％ ,t =16.124, 8.773 and 14.076,P all ＜0.01).(3) RT-qPCR and Western blotting showed that the CASZ1 mRNA significantly increased in experimental group compared with control group (0.56 ±0.17 vs.1.00±0.26, 0.32 ±0.29 vs.0.58 ±0.14,0.14 ±0.22 vs.0.56 ±0.15,t =3.890, 3.303 and 2.388, P ＜ 0.05 for all comparisons), and the protein expression of CASZ1 also significantly increased in experimental group compared with control group (0.65 ± 0.02 vs.0.78 ± 0.23, 0.46 ± 0.03 vs.0.75 ±0.05, 0.34 ±0.22 vs.0.75 ±0.04, t =6.200 and 10.485 and 14.998, P ＜0.05 for all comparisons).(4)The protein level of CASZ1 in experimental group was positively correlated with capillary density (r =0.519, P ＜0.01).Conclusion CASZ1 is involved in the whole process of newborn rats BPD and may be linked to pulmonary microvascular dysplasia.
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