高体积分数氧对小分子干扰RNA介导肺表面活性蛋白A基因沉默后A549细胞的影响

Effect of hyperoxia on A549 cells suppressed with surfactant protein-A suppressed by small RNA interference- mediated gene silencing

目的探索小分子干扰RNA（SiRNA）沉默A549细胞肺表面活性蛋白A（SP-A）基因后高体积分数氧（高氧）对其的影响，探讨SP-A蛋白在高氧肺损伤中的作用。方法体外传代培养A549细胞，将细胞随机分为沉默组、对照组。以Lipofectamine2000转染特异性SiRNASP-A转入A549细胞，持续置于高氧（950mlVLO2和50mL／LCO2）中，分别于培养48h、72h、96h提取A549细胞的总蛋白，收集培养上清。采用Western-blot检测SP-A蛋白的表达，噻唑兰（MTF）检测细胞增殖能力，硫代巴比妥酸（TAB）比色法检测培养基上清中丙二醛（MDA）水平。结果序列特异性SiRNA可显著沉默SP-A基因表达，其中SiRNASP-A2的沉默效率最高（97％）。高氧干预下，与对照组相比，沉默组A549细胞SP-A蛋白表达在高氧48h、72h时均显著减少（P均〈0．05），沉默组A549细胞增殖能力在高氧48h、72h、96h均受到明显抑制（P均〈0．05）。但2组问培养基中MDA水平未见明显差异（P〉0．05）。结论SP-A基因沉默后A549细胞抗高氧能力下降，表明SP-A可能在高氧肺损伤过程起保护作用。

Objective To explore the effect of hyperoxia on A549 cells suppressed with surfactant protein A （SP-A） suppressed by small interference RNA（SiRNA）-mediated gene silencing, and discuss the function of SP-A in hyperoxic lung injm＇y. Methods A549 cells were gained by serial sub cultivation in vitro and randomly divided into 2 groups, silenced of SP-A group and the control group. A549 cells were transfected with synthetic SP-A sequence-specific SiRNA by Lipofectamine 2000, continuously exposed to hyperoxia（950 mL/L 02 ,50 mL/L CO2 ）. After exposure to hy- peroxia for 48 hours ,72 hours and 96 hours ,total protein and culture supernatant were gained. SP-A protein was detec- ted by Western-blot, the capacity of proliferation was detected by methyl thiazolyl tetrazolium, and thiobarbituric acid colorimetric method was used to detect the malondialdehyde（MDA） in culture supernatant. Results Sequence-specific SiRNA targeted SP-A2 and significantly down-regulated its expression in A549 cells. Compared with the control group in hyperoxia, the expression of SP-A significantly decreased after 48 hours ,72 hours in the silenced group（ all P 〈 0.05 ） , and the capacity of proliferation in A549 cells silenced by SP-A were obviously decreased after 48 hours ,72 hours and 96 hours （ all P 〈 0.05 ）. But there was no significant difference in the MDA in culture supernatant between 2 groups （ all P 〉 0. 05 ）. Conclusions The capacity of resisting hyperoxia decreased in A549 cells silenced by SP-A, which indi- cates that SP-A can protect hyperoxic lung injury.