STAT1反义寡核苷酸雾化吸入转染博莱霉素致肺纤维化大鼠模型的建立与鉴定  

THE ESTABLISHMENT AND IDENTIFICATION OF AEROSOLIZED STAT1 ANTISENSE OLIGODEOXYNUCLEOTIDES TRANSFECTED TO BLEOMYCIN-INDUCED PULMONARY FIBROSIS RAT

作　　者：范贤明[1] 周玲[2] 王文军[1] 曾鸣[1] 朱晨[1] 徐萧洪[1] 湛晓勤[1] 

机构地区：[1]泸州医学院附属医院呼吸内科,四川泸州646000 [2]自贡市第一人民医院呼吸内科

出　　处：《泸州医学院学报》2008年第3期237-243,共7页Journal of Luzhou Medical College

基　　金：国家自然科学基金资助项目(30570814);四川省教育厅资助项目(05011)

摘　　要：目的:探讨信号转导和转录活化因子1(STAT1)反义寡核苷酸(ASON)雾化吸入体内转染博莱霉素(BLM)致大鼠肺纤维化的可行性并给予鉴定。方法:取Wistar大鼠20只,气管内灌注BLM7天后,将大鼠随机分为生理盐水(NS)组、脂质体(liposome,LP)组、ASON组、5’端异硫氰酸荧光素(FITC)标记的STAT1-ASON(FITC-ASON)组各5只。FITC-ASON组大鼠于气管内灌注BLM后第7天,雾化吸入脂质体包埋全硫代磷酸修饰并5’端FITC标记的STAT1-ASON后6小时处死大鼠,通过支气管肺泡灌洗收集细胞,用荧光显微镜观察灌洗液细胞和肺组织内STAT1-ASON的分布。NS组、LP组、ASON组3组大鼠则于气管内灌注BLM后第7、9、11、13天分别雾化吸入生理盐水、脂质体、脂质体包埋的STAT1-ASON复合物,3组大鼠于第14天处死,通过支气管肺泡灌洗收集肺泡巨噬细胞(AM),用实时荧光定量聚合酶联反应(FQ-PCR)检测AM中STAT1、ICAM-1mRNA的表达;用Western-blot检测AM中STAT1、ICAM-1蛋白的表达;并检测大鼠血清肝功能指标丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)和肾功能指标尿素氮(BUN)、肌酐(Scr)。结果:大鼠雾化吸入5’端FITC标记的STAT1-ASON后,其支气管肺泡灌洗液细胞和肺组织中可见诱发的黄绿色荧光且主要位于AM内;NS组AM中STAT1、ICAM-1mRNA和蛋白表达水平(1.003±0.116、1.008±0.114;0.66±0.09、0.72±0.10)与LP组(1.063±0.121、1.000±0.157;0.68±0.11、0.65±0.11)比较差异无统计学意义(P>0.05);ASON组(0.267±0.036、0.269±0.042;0.31±0.09、0.38±0.07)与NS组及LP组比较,AM中STAT1、ICAM-1mRNA和蛋白表达水平均显著下降(P<0.05);ASON组、NS组及LP组3组肝肾功能指标两两比较差异无统计学意义(P>0.05)。结论:脂质体包裹的STAT1-ASON雾化吸入能够到达AM和肺组织内,用于体内实验是安全的;STAT1-ASON体内转染后,能抑制AM中STAT1、ICAM-1mRNA蛋白的表达,说明STAT1-ASON雾化吸入体内转染致肺纤维化动物模型是可行的�Objective：To evaluate the feasibility of transfecting signal transducer and activator of transcription 1 （STAT1） antisense oligodeoxynucleotides （ASON） for bleomycin（BLM）-induced pulmonary fibrosis rat by aerosolization in vivo. Methods：Twenty Wistar rats were intratracheally instilled with BLM, and then randomly divided into 4 groups： normal saline （NS） group; liposome （LP） group; ASON group; ASON labeled at the 5′ -end with fluorescein isothiocyanate （FITC-ASON） group. FITC-ASON group was aerosolized with phosphorothioated STAT1-ASON labeled at the 5′-end with FITC/liposome complexes at day 7 after rats were intratracheally instilled with BLM, and then rats were sacrificed at 6 hours after aerosolization. Bronchoalveolar lavage （BAL） was performed to obtain cells. Cells and the lung tissue were observed in fluorescence microscope. NS group,LP group and ASON group were aerosolized NS,liposome and STAT1-ASON/liposome complexes respectively at days 7,9,11,13 after rats were intratracheally instilled with BLM. All rats were sacrificed at day 14. BAL was performed to obtain alveolar macrophage （AM）. The expression of STATl,intercellular adhesion molecular-1 （ICAM-1） messenger ribonucleic acid （mRNA） and protein in AM were detected by real time fluorescent quantitative polymerase chain reaction （FQ-PCR） and Western-blot respectively. At the same time, parameters of liver and kidney function were detected in serum of rats. Results：Induced fluorescence was seen in cells of BAL fluid and lung tissue in BLM-induced pulmonary fibosis rats through fluorescence microscope, and it mainly existed in AM. There was no statistical difference in the expression of STAT1 ,ICAM-1 mRNA and protein in AM between NS group （ 1.003±0.116,1.008±0.114; 0.66±0.09,0.72±0.10） and LP group （ 1.063±0.121,1.000± 0.157;0.68±0.11,0.65±0.11 ） （P〉0.05）. Compared with NS group or LP group, the expression of STAT1,ICAM-1 mRNA and protein in AM significantly decreased in ASON

关 键 词：肺纤维化 STAT1 反义寡核苷酸 雾化吸入 

分 类 号：R563[医药卫生—呼吸系统]