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2008年第31卷第3期 目录
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*
范贤明，周 玲1，王文军，曾 鸣，朱 晨，徐萧洪，湛晓勤
（泸州医学院附属医院呼吸内科，四川泸州 646000；1自贡市第一人民医院呼吸内科）
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BLM 7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-1
mRNA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-1
mRNA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-1
mRNA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-1 mRNA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。
关键词 肺纤维化；STAT1；反义寡核苷酸；雾化吸入
*基金项目：国家自然科学基金资助项目（30570814），四川省教育厅资助项目（05011）
THE ESTABLISHMENT
AND IDENTIFICATION OF AEROSOLIZED STAT1 ANTISENSE OLIGODEOXYNUCLEOTIDES
TRANSFECTED TO BLEOMYCIN-INDUCED PULMONARY FIBROSIS RAT
Fan Xianming,et al
Department of Respiratory Medicine,the Affiliated Hospital of
Luzhou Medical College
Abstract 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 STAT1,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 group（0.267±0.036、0.269±0.042；0.31±0.09、0.38±0.07）（P＜0.05）.
There was no statistical difference in the concentration of
ALT,AST,BUN and Scr in serum of rats among NS group,LP group
and ASON group (P>0.05). Conclusion:Aerosolized STAT1-ASON
encapsulated by liposome could enter into AM and lung tissue,
and there was no toxic side effect of liver and kidney in BLM-induced
pulmonary fibrosis rats. Transfecting STAT1-ASON in vivo could
inhibit mRNA and protein expression of STAT1,ICAM-1 in AM of
pulmonary fibrosis rats. Aerosolized STAT1-ASON to transfect
BLM-induced pulmonary fibosis rats is feasible.
Key words Pulmonary fibrosis; Signal transducer
and activator of transcription 1 (STAT1); Antisense oligodeoxynucleotides
(ASON); Aerosolization
