高浓度氧暴露对早产鼠肺泡Ⅱ型上皮细胞还原型烟酰胺腺嘌呤二核苷酸表达的影响

Effect of Hyperoxia on Expression of Reduced Form of Nicotinamide-Adenine Dinucleotid-Dehydrogenase Subunit 4 of Alveolar Epithelial Cells Type Ⅱ from Preterm Rats

目的探讨高浓度氧(高氧)暴露对早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)还原型烟酰胺腺嘌呤二核苷酸(NADH)-脱氢酶亚基4(ND4)表达的影响。方法原代培养早产Sprague-Dawley(SD)大鼠AECⅡ,待其在含50mL/L二氧化碳(CO2)培养箱中生长至接近融合状态时随机分为高氧组和空气组。高氧组持续暴露于常压高浓度氧中,氧体积分数>900mL/L,CO2体积分数为50mL/L;空气组仍置于含50mL/L CO2培养箱中。二组分别于高氧或空气暴露6、12、24h提取细胞RNA,采取半定量反转录聚合酶链反应(RT-PCR)测定ND4 mRNA的表达;采用免疫细胞化学染色法检测细胞爬片ND4蛋白的表达。结果与空气组比较,免疫细胞化学结果显示高氧暴露6、12h ND4的表达无显著性差异(Pa>0.05),高氧暴露24h ND4的表达显著减弱(P<0.05);RT-PCR检测显示高氧暴露6h ND4 mRNA表达无显著性差异(P>0.05),高氧暴露12、24h ND4 mRNA表达显著减弱(Pa<0.05)。结论高氧暴露下调早产SD大鼠AECⅡ ND4 mRNA和蛋白水平的表达,这种改变可能参与高氧肺损伤的发病过程。

Objective To explore the effect of hyperoxia on the expression of reduced form of nicotinamide - adenine dinueleotid（NADH） - dehydrogenase subunit 4 （NIM） of alveolar epithelial cells type Ⅱ （ AEC Ⅱ ） from preterm SD rats. Methods Cells were gained by primary euhure from SD rat lungs. When approaehing to the condition of confluence in carbon dioxide（ CO2 ） euhure chamber（ 37℃ ,50 mL/L CO2 ） , the cells were randomly divided into air group and hyperoxia group. Hyperoxia group was continuously exposed to oxygen（ oxygen 〉 900 mL/L, CO2 = 50 mL/L）. While air group still in specific room air（ CO2 = 50 mL/L）. After exposure to oxygen or room air for 6,12,24 hours, those cells＇ RNA was extracted and ND4 mRNA was detected by reverse transcription polymerase chain reaction（ RT- PCR）. Immunocytoehemistry was used to detect the expression of ND4 protein in cell slides. Results Compared with air group,the expression of ND4 detected by immune stainning did not change significantly after exposure to oxygen for 6,12 hours（Pa 〉 0.05） ,but decreased significantly after exposure to oxygen for 24 hours （ P 〈 0.05 ） ; ND4 mRNA expression did not ehange significantly after exposure to oxygen for 6 hours（ P 〉 0.05 ） , but decreased significantly after exposure to oxygen for 12 hours and 24 hours（ Pa 〈 0.05 ）. Conclusion Continuous exposure to hyperoxia down - regulates the expression of NADH- dehydrogenase subunit 4 ,which participates in the process of lung injury induced by hyperoxia.