摘要
目的在脓毒症急性肺损伤中探讨miR-98-5p对肺泡巨噬细胞表型分化的影响并初步探究其相关分子机制。方法利用盲肠结扎穿孔(cecal ligation and puncture,CLP)术建立大鼠急性肺损伤(ALI)模型,并将40只SD大鼠分为4组(n=10):假手术(Sham)组,脓毒症组(以下简称CLP),CLP^+agomir NC组(CLP^+agomir NC),CLP^+miR-98-5p agomir组(CLP^+miR-98-5p agomir)。在CLP术后24 h处死各组大鼠并收集肺泡灌洗液(BALF)及双肺组织,利用HE染色、ELISA及检测肺组织干湿比重(W/T)来探究在大鼠体内过表达miR-98-5p对脓毒症介导的ALI的肺组织结构、肺泡灌洗液(BALF)中炎性因子的表达及肺部含水量的影响;分离大鼠原代肺泡巨噬细胞(AMs),细胞流式检测过表达miR-98-5p对ALI大鼠AMs的M1、M2表型分化影响;免疫磁珠分选F4/80^+AMs,利用RT-PCR检测M1、M2表型特征分子标记基因iNOS、CD206、Arg1的表达水平;生物信息学筛选TRAF6作为miR-98-5p的潜在靶基因,利用双荧光素酶基因报告实验进行检测;利用RT-PCR检测过表达miR-98-5p后的ALI大鼠AMs中miR-98-5p与TRAF6 mRNA的表达;应用细胞免疫化学法与Western blot实验在F4/80^+AMs中检测TRAF6与TLR4/MyD88/NF-κB信号通路中TLR4、MyD88、p65、p-p65蛋白的表达。结果HE染色、ELISA及肺组织干湿比重(W/T)的检测结果表明,在脓毒症大鼠ALI模型中,过表达miR-98-5p可明显缓解ALI大鼠肺部病理学改变,降低BALF中促炎因子IL-6、IL-1β、TNF-α的分泌水平,增加抗炎因子IL-10的分泌,并减少ALI肺部含水量;细胞流式结果显示过表达miR-98-5p可诱导ALI大鼠AMs向M2表型分化,抑制其向M1表型分化;在F4/80^+AMs中,RT-PCR实验结果显示,过表达miR-98-5p可抑制M1表型特征分子iNOS mRNA表达水平,而促进M2型特征分子CD206、Arg1表达水平;双荧光素酶基因报告实验结果显示,在大鼠AMs中miR-98-5p可靶向调控ARTF6 mRNA的表达。细胞免疫化学法与Western blot实验结果显示,过表达miR-98-5p明显�
This study was performed to investigate the effect of microRNA-98-5 p on acute lung injury in sepsis and its molecular mechanism.The rat model of acute lung injury(ALI)was established by cecal ligation and puncture.Forty SD rats were divided into four groups(n=10):sham group,sepsis group(CLP),CLP^+agomir NC group(CLP^+agomir NC)and CLP^+microRNA-98-5 p agomir group(CLP^+miR-98-5 p agomir).At 24 hours after CLP modeling,rats in each group were sacrificed to collect alveolar lavage fluid(BALF)and bilateral lung tissues.HE staining,ELISA and W/T were respectively used to investigate the effects of miR-98-5 p over-expression on pulmonary tissue structure,the expression of inflammatory factors in alveolar lavage fluid(BALF)and pulmonary water content in rats with sepsismediated ALI.Rat primary alveolar macrophages(AMs)were isolated and the effects of over-expression of miR-98-5 p on the phenotypic differentiation of M1 and M2 in ALI rats were detected by flow cytometry.Immunomagnetic beads were used to isolate F4/80^+AMs,and RT-PCR was used to detect the expression levels of M1 and M2 phenotypic marker genes iNOS,CD206 and Arg1.screened TRAF6 was screened out by bioinformatics,and used as a potential target gene for microRNA-98-5 p detection by double luciferase gene reporting assay.RT-PCR was used to detect the expression of miR-98-5 p and TRAF6 in AMs of ALI rats after overexpression of miR-98-5 p.Cytoimmunochemistry and Western blot were used to detect the expressions of TLR4,MyD88,p65 and p-p65 proteins in TRAF6 and TLR4/MyD88/NF-kappa B signaling pathways in F4/80^+AMs.The results of HE staining,ELISA and W/T of lung tissue showed that over-expression of miR-98-5 p could significantly alleviate the pathological changes of ALI rats’lungs,decrease the secretion levels of pro-inflammatory factors IL-6,IL-1 beta and TNF-alpha in BALF,increase the secretion of anti-inflammatory factor IL-10,and decrease the secretion of anti-inflammatory factor IL-10 and water content in ALI rats’lungs.Flow cytometry showed that o
作者
唐晋
周靖
陈雪梅
吴薇
彭超华
TANG Jin;ZHOU Jing;CHEN Xuemei;WU Wei;PENG Chaohua(Intensive Care Unit,West China-Guang’an Hospital,Sichuan University,Guang'an 638000,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2020年第8期645-654,共10页
Immunological Journal
基金
四川省医学科研青年创新课题(016031)。
