摘要
为研究microRNA-124-3p(miR-124-3p)对H1N1亚型猪流感病毒(swine influenza virus,SIV)感染小鼠所致肺损伤的调控作用,本试验构建miR-124-3p腺病毒表达载体,通过小鼠尾部静脉注射法构建miR-124-3p差异表达小鼠模型,试验分3组:过表达组、抑制组和对照组。48 h后,各组小鼠鼻腔接种H1N1亚型SIV,每只105 EID50(50μL)。连续观察14 d,计算小鼠平均体重变化率、观察病理切片并测定相关炎症因子IL-1β、TNF-α和IL-6 mRNA相对表达量。结果显示,已成功将pre-miR序列及其sponge序列插入腺病毒的穿梭质粒,并将其共转染293A细胞。实时荧光定量PCR检测证实,与对照组相比,过表达组和抑制组小鼠黑色素瘤细胞miR-124-3p表达水平分别极显著升高(P<0.01)和显著降低(P<0.05),表明成功构建腺病毒表达载体。过表达组、抑制组和对照组小鼠体重变化率分别为-5.5%、-12.4%和-8.6%。抑制组和对照组均可见肺泡壁增厚,其间有多量淋巴细胞浸润,部分肺泡内出现纤维蛋白渗出,且抑制组病理变化更为严重,肺泡中还有大量的红细胞浸润;而过表达组仅有少量的淋巴细胞浸润,肺脏组织较正常。与对照组相比,过表达组检测的炎症因子IL-1β、TNF-α和IL-6 mRNA表达水平均显著降低(P<0.05);抑制组炎症相关炎症因子mRNA表达水平均显著升高(P<0.05)。本试验结果表明,miR-124-3p对H1N1亚型SIV感染小鼠所致的肺脏炎症因子的表达具有抑制作用,同时能减轻肺脏病理损伤。
In order to study the regulatory effect of microRNA-124-3p(miR-124-3p)on lung injury caused by H1N1 subtype swine influenza virus(SIV)in mice,the expression vector of miR-124-3p adenovirus was constructed,and the miR-124-3p differentially expressed mouse models were induced by intravenous injection in the tail of mice which were divided it into three groups(overexpression,inhibition and control groups).48 h later,mice in each group were inoculated with H1N1 subtype SIV in the nasal cavity,with 105 EID50(50μL)per mouse.The average body weight change rate,pathological section observation and relative mRNA expression level of related inflammatory factors IL-1β,TNF-αand IL-6 in mice were observed for 14 consecutive days.The results showed that the pre-miR sequence and its sponge sequence had been successfully inserted into the shuttle plasmid of adenovirus,and co-transfected into 293A cells.Real-time PCR detection confirmed that compared with control group,the expression level of miR-124-3p in overexpression and inhibition groups were extremely significantly increased(P<0.01)and significantly decreased(P<0.05),respectively,indicating that the adenovirus expression vectors were successfully constructed.The rates of weight change of overexpression,inhibition and control groups were-5.5%,-12.4%and-8.6%,respectively.The alveolar wall of inhibition and control groups were thickened,in which there were a lot of lymphocyte infiltration,some of the alveoli showed fibrin exudation,and the pathological changes were more serious in the inhibition group,there were a lot of RBC infiltration in alveoli.There were only a little lymphocyte infiltration in overexpression group,and the lung tissue was normal.Compared with control group,the mRNA expression levels of IL-1β,TNF-αand IL-6 in overexpression group were significantly decreased(P<0.05);The mRNA expression levels of inflammatory factors in inhibition group were significantly increased(P<0.05).The results showed that miR-124-3p inhibited the expression of pulmonary inflammatory factors induced by H1N1 subtype SIV in mice,and also alleviated pathological injury of lung.
作者
黄良宗
张海龙
颜广智
谢博
陈盛楠
邓汝森
顾万军
HUANG Liangzong;ZHANG Hailong;YAN Guangzhi;XIE Bo;CHEN Shengnan;DENG Rusen;GU Wanjun(School of Life Science and Engineering,Foshan University,Foshan 528231,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第10期3049-3057,共9页
China Animal Husbandry & Veterinary Medicine
基金
广东省自然科学基金项目(2017A030310612)
广东省教育厅预防兽医学重点实验室项目(2014KTSPT037)
佛山科学技术学院研究生自由探索基金项目(2019ZYTS001)。
