RNA干扰下调Caveolin-1基因对大鼠肺水肿的影响

Effects of RNA interference-induced Caveolin-1 downregulation on lung edema of rats

目的观察RNA干扰（RNAi）下调大鼠肺脏Caveolin-1基因表达对其肺脏微血管通透性的影响。方法164只SD大鼠随机分为实验组和对照组，以阳离子脂质体为载体，将生物合成的Caveolin-1小干扰RNA（siRNA）及Caveolin-1 siRNA阴性对照链按0．4、0．8、1．2mg／kg 3个剂量梯度分别注入大鼠双侧肺脏，注射后24、48、72h 3个时间点以Western blot及免疫组织化学（IHC）检测肺脏Caveolin-1蛋白的表达，逆转录。聚合酶链反应（RT-PCR）检测Caveolin-1基因表达，Westernblot检测水通道蛋白（AQP）-1的表达。胸部透视观察肺水肿情况。以1．0ml／kg标准将1％伊文氏兰经尾静脉注射人体内后，取肺中叶浸浴甲酰胺中，分光光度计检测620nm处吸光度（A值）。结果实验组72h时Caveolin．1基因沉默率约为85％，实验组AQP-1表达变化不明显；对照组3个浓度梯度间Caveolin-1表达差异无统计学意义（P〉0．05）。胸透显示实验组肺叶密度增高，在3个浓度梯度分别转染72h后，甲酰胺A值较对照组明显增高（P〈0．01）。结论Caveolin-1蛋白是调控肺水肿形成的重要因素，且其调控作用不通过AQP-1途径。

Objective To explore the effects of down-regulation of Caveolin-1 gene expression on the permeability of lung capillaries. Methods Small interfering RNA （siRNA） was used to knock-clown Caveolin-1 expression in rat lungs. A single-stranded siRNA construct was designed, and another sequence was used as a control construct. Pulmonary injection of siRNA against Caveolin-1 mRNA incorporated in liposomes with three dosage gradients of 0. 4, 0. 8 and 1.2 mg/kg. The protein expression of Caveolin-1 was detected by Western blotting and immunohistochemistry. Reverse transcription-polymerase chain reaction （RT-PCR） demonstrated the expression of eaveolin-1 expression. The expression of Aquaporin （AQP）-1 protein was detected by using Western blotting. Chest fluoroscopy showed the degree of lung edema. 1% of Evans blue was injected through vena caudalis with a dosage of 1.0 mL/kg, and then lobus medius pulmonis was dissected and immersed in formamide. Results At 72nd h in experimental group, the gene silencing of Caveolin-1 siRNA was about 85%. There was slight difference in the Caveolin-1 expression in the control group with the three dosage gradients. Caveolin-1 siRNA had little influence over the AQP-1 protein level. Under the chest fluoroscopy, density of pulmonary lobes was increased. In comparison with the control group, the absorbance values of formamide in experimental group were significantly increased （ P 〈 0. 01 ）. Conclusion Caveolin-1 may play an important role in the process of lung edema, which was AQP- 1 independent.