microRNA-7敲减对内毒素诱导小鼠急性肺损伤的影响

Effect of microRNA-7 knockdown on pathology of Enterotoxin-induced murine acute lung injury

目的:观察microRNA-7(miR-7)敲减对内毒素诱导小鼠急性肺损伤的影响并探讨其意义。方法:利用脂多糖(LPS)腹腔注射(10 mg/kg体重)野生型(Wild type,WT)小鼠和miR-7基因敲减(Knockdown,KD)小鼠建立急性肺损伤(Acute lung injury,ALI)模型;HE染色观察肺组织病理学变化;计算肺泡灌洗液(BAL)中的细胞总数;Real-time PCR检测肺组织炎症相关因子表达变化;流式细胞术(FACS)进一步检测BAL中固有免疫细胞γδT细胞、F4/80巨噬细胞(Macrophages,Mφ)和适应性免疫细胞CD4^+T细胞和CD8^+T细胞的比例和绝对数变化;FACS检测CD4^+T细胞活化相关分子CD62L和CD69的表达水平。结果:相对野生型(Wild type,WT)小鼠,HE染色显示miR-7KD小鼠的肺组织小血管充血和炎性细胞浸润明显减少,病理性损伤明显减轻;Real-time PCR结果显示促炎性细胞因子IL-6的表达水平显著降低(P<0.01),而抗炎性细胞因子IL-4、TGF-β的表达水平明显增加(P<0.05);同时,BAL中总细胞数显著减少(P<0.05);FACS检测进一步显示miR-7KD小鼠的BAL中F4/80^+Mφ细胞的比例及绝对数均明显下调(P<0.05),而γδT细胞的比例上调(P<0.05),但其细胞绝对数无差异(P>0.05);BAL中CD4^+T细胞和CD8^+T细胞的比例及其绝对数均显著降低(P<0.05);最后CD4^+T细胞活化膜分子CD62L的表达水平明显上调(P<0.05),而CD69的表达水平显著下调(P<0.05)。结论:miR-7敲减可减轻ALI模型小鼠的肺部损伤,提示其可能在ALI发生中发挥了重要调控作用。

Objective：To detect the effect of microRNA-7（miR-7） knockdown on pathology in murine acute lung injury（ALI） model,and preliminarily explore its significance.Methods：Murine ALI model was performed by intraperitoneal injection of Lipopolysaccharide（LPS）（10 mg/kg） into miR-7KD mice and wild-type（wild type,WT） mice respectively.Then,the pathologic injury of lung tissue were observed by HE staining.And total cell count of bronchoalveolarlavage（BAL） was calculated.The relative expression of related cytokines in lung tissue was analyzed by Real-time PCR assay.Furthermore,the changes on proportion of innate immune cells（γδT cell and F4/80 macrophages cell） and adaptive immune cell（CD4~＋T cell and CD8~＋T cell） were analyzed by FACS.Meanwhile,the expression of CD62 L and CD69,as well as the absolute number,in CD4~＋T cell were also analyzed.Results：Compared with WT mice,pathological damage in lung tissues was significantly alleviated in miR-7KD mice.Real-time PCR analysis showed that the relative expression of IL-6 was obviously reduced（P 0.01）,conversely,relative expression of IL-4 and TGF-β were obviously increased（P 0.05）.Furthermore,the total cell number in BAL also reduced significantly（P〈0.05）.Importantly,FACS analysis showed that the proportion and the absolute number of F4/80~＋Mφ cells obviously reduced（P〈0.05）;however,the proportion of γδT cells increased（P 0.05）.Moreover,the proportion and the absolute number of CD4~＋T cells and CD8~＋T cells were significantly reduced（P〈0.05）.Finally,the proportion and the absolute number of CD62L~＋in CD4~＋T cells were upregulated vigorously,contrastly,the proportion and the absolute number of CD69~＋in CD4~＋T cells were notably up-regulated（P〈0.05）.Conclusion：miR-7 defeciency could significantly ameliorate the pathology of murine ALI,suggesting that it may play an important regulatory role in the development of ALI.