miR-155反义寡核苷酸对内毒素诱导急性肺损伤小鼠的保护作用  

Protective effect of microRNA-155 antisense oligonucleotid on lipopolysaccharide-induced acute lung injury in mice

作　　者：唐瑾[1] 谢旺[2] 程婷婷[2] 王凯玲[2] 顾霞[2] 张倩 郭忠良[2] Tang Jin;Xie Wang;Cheng Tingting;Wang Kailing;Gu Xia;Zhang Qian;Guo Zhongliang(Department of Central ICU,East Hospital,Tongji University School of Medicine,Shanghai 200123,China;Department of Respiration,East Hospital,Tongji University School of Medicine,Shanghai 200123,China)

机构地区：[1]同济大学附属东方医院中心ICU,上海200123 [2]同济大学附属东方医院呼吸内科,上海200123

出　　处：《中华危重病急救医学》2018年第8期743-747,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81370174）

摘　　要：目的 通过建立微小RNA（miRNA）反义寡核苷酸（ASO）慢病毒表达载体,探讨miR-155 ASO对急性肺损伤（ALI）小鼠的保护作用.方法 设计并合成miR-155 ASO,使用BamHⅠ和NheⅠ双酶切后连接产生慢病毒表达载体,聚合酶链反应（PCR）筛选阳性克隆,测序并测定病毒滴度.按随机数字表法将54只4～6周龄的雄性BALB/c小鼠平均分为3组,均经腹腔注射脂多糖（LPS）10 mg/kg制备ALI动物模型.3组分别于制模前24 h经尾静脉注射含1×108/mL pmiR-155-ASO病毒的磷酸盐缓冲液（PBS）200μL（pmiR-155-ASO组）,或含1×108/mL pSMPUW-miR-GFP空载体病毒的PBS 200μL（pmiR-cont组）,或等量PBS（PBS组）.每组中10只小鼠用于观察7 d存活率;剩余8只小鼠于制模后取血标本及肺组织,用酶联免疫吸附试验（ELISA）测定血清炎性因子水平;用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测肺组织miR-155表达;镜下观察肺组织病理学改变及巨噬细胞分布.结果 pmiR-cont组与PBS组各指标比较差异均无统计学意义.pmiR-155-ASO组肺组织中miR-155成熟体的表达水平较pmiR-cont组明显降低（2-ΔΔCt：4.92±0.72比15.38±0.60,P〈0.05）.与pmiR-cont组比较：给予miR-155ASO预处理后ALI小鼠损伤程度明显改善,7 d存活率显著提高（72.1％比61.9％,P〈0.05）;肺大体观察显示肺组织充血明显减轻,肺湿/干重（W/D）比值明显下降（4.50±0.13比5.64±0.61,P〈0.05）;苏木素-伊红（HE）染色显示肺组织炎性细胞浸润减少,免疫荧光法检测显示肺组织巨噬细胞浸润数量明显减少;血清中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平明显降低〔TNF-α（ng/L）：379.8±48.9比495.9±33.3,IL-6（ng/L）：262.3±61.8比355.4±22.6,均P〈0.05〕,而IL-10水平无明显改变（ng/L：143.6±32.5比140.4±22.3,P〉0.05）.结论 miR-155 ASO具有抑制LPS诱导ALI小鼠炎症反应、改善预后的作用. ObjectiveTo investigate the protective effect of microRNA-155 （miR-155） antisense oligonucleotid （ASO） on acute lung injury （ALI） mice by establishing a lentiviral expression vector of ASO of miRNA.Methods miR-155 antisense oligonucleotides amplified by polymerase chain reaction （PCR） from genomic, using BamH Ⅰ and Nhe Ⅰ double digestion, ligated into lentiviral expression vector. Sequence and virus titer were measured. According to the random number table method, 54 male BALB/c mice of 4-6 weeks old were divided into three groups. ALI animal models were prepared by intraperitoneal injection of 10 mg/kg lipopolysaccharide （LPS）. The three groups were injected with 200μL phosphate buffered saline （PBS） containing 1×108/mL pmiR-155-ASO virus （pmiR-155-ASO group） or 200μL PBS containing 1×108/mL pSMPUW-miR-GFP empty virus （pmiR-cont group） or the same amount of PBS （PBS group） at 24 hours before the molding. Ten mice in each group were used to observe the 7-day survival rate. Blood samples and lung tissues of the remaining 8 mice were harvested after the model was established, and the levels of serum inflammatory cytokines were determined by enzyme linked immunosorbent assay （ELISA）; the expression of miR-155 in lung tissue was detected by real-time reverse transcription-polymerase chain reaction （RT-PCR）; histopathological changes of lung and distribution of macrophages were observed under microscope.Results There was no significant difference in each index between pmiR-cont group and PBS group. The mature miR-155 expression in lung tissue in pmiR-155-ASO group was significantly lower than that in pmiR-cont group （2-ΔΔCt： 4.92±0.72 vs. 15.38±0.60,P 〈 0.05）. Compared with pmiR-cont group, the injury degree of ALI mice after pretreatment with miR-155ASO was significantly improved, and the 7-day survival rate was significantly increased （72.1% vs. 61.9%,P 〈 0.05 ）; gross lung observation showed that congestion in lung tissue was significantly re

关 键 词：急性肺损伤 炎症反应 微小RNA-155 反义寡核苷酸 

分 类 号：R563[医药卫生—呼吸系统]