慢病毒介导TGF-β1 siRNA对高体积分数氧致新生小鼠肺损伤的影响  

Influence of TGF-β1 siRNA mediated by lentivirus vector on hyperoxia-induced lung injury in neonatal mice

作　　者：段江[1,2] 陈洪清[2] 李然[2] 尹晓娟[2] 封志纯北京军区总医院附属八一儿童医院 

机构地区：[1]南方医科大学附属珠江医院儿科,广东广州510280 [2]北京军区总医院附属八一儿童医院,北京100700

出　　处：《第四军医大学学报》2009年第9期785-788,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30772036)

摘　　要：目的:探讨以慢病毒介导的小分子干扰RNA技术(siRNA)沉默转化生长因子β1(TGF-β1)基因对高体积分数氧致新生小鼠肺损伤的影响.方法:将100只新生昆明小鼠随机分为高氧组、干扰组、阴性干扰组、空气组4组,每组动物25只.分别构建含TGF-β1短发卡RNA(shRNA)及阴性对照序列的PRNAT-u6.2载体,慢病毒包装.于小鼠出生后3d时将两类慢病毒载体以鼻腔吸入法分别导入干扰组和阴性干扰组小鼠肺内.出生后4～14d时将高氧组、干扰组、阴性干扰组小鼠置于含600mL/LO2的氧箱内饲养.空气组仅置空气下饲养.出生后4,7,14,21,28d时分别随机杀取各组5只动物并取左上肺制作石蜡切片,进行组织形态学变化观察,测定放射状肺泡计数(RAC)和肺泡平均截距(MLI).右肺组织以RT-PCR,Western Blot法分别检测TGF-β1 mRNA和蛋白表达水平.结果:经测序和双酶切鉴定,表达TGF-β1 shRNA的慢病毒载体构建成功;干扰组肺组织TGF-β1 mRNA水平在小鼠出生后4d即明显低于其他3组(P<0.05);出生后7,14,21,28d时干扰组TGF-β1 mRNA和蛋白表达水平均低于高氧组和阴性干扰组,但高于空气组(P均<0.05).出生后7,14,21,28d干扰组MLI低于高氧组和阴性干扰组而高于空气组(P均<0.05).RAC高于高氧组和阴性干扰组而低于空气组,差异均具有统计学意义(P<0.05).高氧组与阴性干扰组在各时间点上述各项指标的差异无统计学意义.结论:慢病毒载体介导的TGF-β1小分子干扰RNA对高体积分数氧吸入所致新生小鼠肺损伤具有保护作用.AIM： To explore the influence of knock-down TGF- β1 gene by lentivirus vector-mediated small interfering RNA （siRNA） on hyperoxia-induced lung injury in neonatal mice. METHODS： One hundred newborn KM mice were randomly divided into hyperoxia group, interference group, negative control group and air group, with 25 mice in each group. PRNAT-u6.2 vectors containing short hairpin （shRNA）of TGF-β1 or negative control sequence were constructed and packaged with lentivirus plasmids. Two types of lentivirus vectors were administered intranasally to lungs of mice in interference group and negative control group respectively at postnatal day 3. Mice in hyperoxia group, interference group and negative control group were exposed to 600 mL/L oxygen from postnatal day 4 to day 14 before resto- ring to room air condition. Mice in air group were exposed to room air condition. Five mice per group were randomly sacrificed and lung tissues were excised at postnatal days 4, 7, ld, 21 and 28 respectively. Left upper lobes were processed for paraffin embedding and sectioning. Mean linear intercept （MLI）and radical alveolar counts （RAC）were counted. Right lobes were excised and frozen for detecting expression of TGF-β1 mRNA and protein by RT-PCR and Western blot methods respectively. RESULTS： Lentivirus vector of TGF-~I shRNA was successfully constructed, which was confirmed by double restriction enzyme digestion and sequencing. Compared with that in the other 3 groups, the expression of TGF-β1 mRNA in lungs decreased significantly in interference group at postnatal day 4 （ P 〈 0.05 ）. At postnatal days 7, 14, 21 and 28, the expression of TGF-f31 mRNA and TGF-β1 protein in lungs of interference group were lower significantly than those in hyperoxia group and negative control group （ P 〈 0.05 ） ,but higher than those in air group（P 〈0.05）. MLI of alveoli de- creased and RAC increased in interference group compared with those in hyperoxia group and negative control group （ P 〈0.05 ）,and

关 键 词：转化生长因子Β1 小分子干扰RNA 高体积分数氧 支气管肺发育不良 小鼠 

分 类 号：R722.6[医药卫生—儿科]