miRNA-200a阻抑小鼠矽肺纤维化的实验研究

Experimental study of the inhibition of miRNA-200a on silica-induced lung fibrosis in mice

目的观察过表达miRNA-200a重组慢病毒Lv-miR-200a在小鼠矽肺模型中对β-catenin的影响及矽肺纤维化的抑制作用,为探讨矽肺的分子机制奠定实验基础。方法将小鼠分为生理盐水组、Si O2模型组(3mg Si O2)、过表达miRNA-200a的重组慢病毒(Si O2+Lv-miR-200a)组和对照慢病毒(Si O2+Lv-NC)组,每组18只。染尘7、14和28d后处死动物。Realtime-PCR检测肺组织中miRNA-200a和β-catenin的表达情况;ELISA法检测肺泡灌洗液中TGF-β1、TNF-α、IL-6和IL-1β细胞因子含量;利用羟脯胺酸试剂盒测定肺组织中羟脯胺酸含量;HE和Masson染色观察肺组织病理变化。结果染尘7、14和28d时,Si O2+Lv-miR-200a组中miRNA-200a的表达明显高于其余三组,β-catenin表达明显低于Si O2模型组和Si O2+Lv-NC组,差异有统计学意义(P<0.05);染尘7d时Si O2+Lv-miR-200a组的肺泡灌洗液中TGF-β1、TNF-α、IL-6和IL-1β的含量比Si O2模型组和Si O2+Lv-NC组明显减少,差异有统计学意义(P<0.05);Si O2+Lv-miR-200a组中的羟脯胺酸含量与Si O2模型组和Si O2+Lv-NC组相比明显减少,差异有统计学意义(P<0.05),病理学观察Si O2+Lv-miR-200a组中肺纤维化程度明显减轻。结论 miR-200a通过调控Wnt/β-catenin信号通路抑制矽肺纤维化的发展。

Objective To obser^^e the effect of recombinant lentivirus Lv-miR-200a on p-catenin and the inhibition of silicosis in silicosis model of mice,and provide clues for exploring the molecular mechanism of silicosis.Methods The mice were divided into normal saline group,Si02 model group(Si02),recombinant lentivirus group overexpressing miR-200a(Si02+Lv-miR-200a)and control lentivirus group(Si02+Lv-NC),18 in each group.Animals were sacrificed at 7,14 and 28 days post-instillation.The expression of miR-200a and p-catenin in lung tissue was detected by Realtime-PCR.The levels of TGF-p1,TNF-a,IL-6 and IL-1p in broncho-alveolar lavage fluid(BALF)were detected by ELISA.The hydroxyproline content in lung tissue was measured by hydroxyproline reagent kit.HE and Masson staining were used to observe the pathological changes of lung tissue.Results The expression of miR-200a was significantly higher than that in the three groups.The expression of p-catenin in Si02+Lv-miR-200a group was significantly lower than that in Si02 model group and Si02+Lv-NC group at 7,14 and 28 days(P<0.05).The levels of TGF-p1,TNF-a,IL-6 and IL-1 p in the Lv-miR-200a group were significantly lower than those in the Si02model group and the Si02+Lv-NC group(P<0.05);and the content of hydroxy-proline significantly decreased(P<0.05),and the degree of pulmonary fibrosis reduced in the Si02+Lv-miR-200a group than in the Si02 model group and the Si02+Lv-NC group.Conclusion miR-200a inhibited the development of silicosis by regulating Wnt/p-catenin signaling pathway.