【作者】 段江；

【导师】 封志纯；

【作者基本信息】 南方医科大学 ， 儿科学， 2009， 博士

【摘要】 支气管肺发育不良（Bronchopulmonary dysplasia,BPD）是早产儿最主要的死亡原因之一。存活的患儿则多残留阻塞性气道疾病,肺动脉高压以及严重的生长发育迟滞。尽管近20年来随着早产儿重症监护技术和肺表面活性物质的运用,早产儿存活率已显著提高,但BPD的发生率却有增无减。目前仍缺乏对BPD的根本性治疗手段,探索其病理机制、寻找更好的治疗手段一直是亟待解决的难题。目前研究认为持续高体积分数氧（高氧）吸入致肺损伤是BPD的明确病因之一,但其病理机制并不清楚。研究发现在BPD支气管肺泡灌洗液或肺组织中转化生长因子β1（Transforming growth factor-β1,TGF-β1）的表达明显升高,但目前缺乏系统的相关研究。慢病毒载体具有感染细胞种类广泛,感染能力稳定,表达时限长的优势,而小分子干扰RNA（Small interfering RNA,siRNA）具有特异沉默目的基因的特点。我们用慢病毒介导的siRNA技术,在新生昆明小鼠高氧吸入前直至吸入后一个月的肺组织发育关键时期,活体内持续抑制TGF-β1的高表达,以研究其对新生小鼠持续高氧吸入所致肺损伤是否具有保护作用。第一章靶向TGF-β1小分子干扰RNA慢病毒载体的构建与鉴定目的以小分子干扰RNA技术构建小鼠TGF-β1-shRNA慢病毒载体,并在肺泡二型上皮细胞（Alveolar epithelial cellsⅡ,AECⅡ）中行体外干扰效率鉴定。方法分别设计TGF-β1-shRNA干扰序列及阴性对照序列,BLAST验证基因同源性。设计的序列经体外退火形成双链寡核苷酸,将其与以BamH1/Xhol酶切后的载体pRNAT-u6.2连接,转化DH5α感受态细胞,小量抽提质粒后测序鉴定。得到的阳性质粒以Xhol和Xbal双酶切,回收其中7010bp条带作为载体。载体PKG-RFP以Xhol和Nhel双酶切,回收其中1262bp作为目的基因片段纯化,以上两片段连接重组,转化DH5α感受态细胞,抽提质粒。以BamH1单酶切鉴定,释放出600bp条带为正确的克隆质粒,即为重组慢病毒载体。将慢病毒包装系统三质粒和重组慢病毒载体混合,导入293T细胞,包装成病毒后,采集病毒上清液,采用系列梯度稀释法,G418药物筛选测定病毒滴度。同法构建阴性干扰慢病毒载体。将包装好的病毒转染AECⅡ,分为干扰组,阴性干扰组和空白对照组3组,每组5复孔,干扰组和阴性干扰组分别加入构建的干扰载体和阴性干扰载体,空白对照组加入等量不含病毒的培养基。转染96h后提取细胞蛋白质,以Western Blot检验TGF-β1蛋白表达验证慢病毒载体干扰效率。并分别取3实验组AECⅡ细胞滴片,以TGF-β1多克隆抗体为一抗,FITC标记的山羊抗兔IgG为二抗,间接免疫荧光法观察TGF-β1表达情况。采集的数据用SPSS13.0统计软件行单因素方差分析,多重比较用SNK法。结果测序结果显示,TGF-β1-shRNA干扰序列成功插入pRNAT-u6.2载体,该重组质粒与PKG-RFP载体重组、转化、氨苄青霉素抗性筛选阳性克隆,酶切验证质粒重组成功。在293T细胞中进行病毒包装,收集上清液,系列梯度稀释法检测病毒悬液的滴度,G418药物筛选,测定慢病毒滴度为5.23×10⁸TU/ml。Western Blot检验TGF-β1蛋白表达,干扰组明显低于阴性干扰组（P＜0.05）和空白对照组（P＜0.05）,抑制效率为73.6%。间接免疫荧光显示阴性干扰组和空白对照组的荧光则呈较强表达,而干扰组的绿色荧光表达则明显减弱。结论成功构建小鼠TGF-β1-shRNA慢病毒载体pU6.2-TGF-RFP-Lenti。其可有效抑制AECⅡTGF-β1蛋白的表达。关键词转化生长因子β1小分子干扰RNA慢病毒载体小鼠第二章活体内沉默TGF-β1基因对小鼠肺泡发育的影响目的:探讨活体内将慢病毒载体导入小鼠肺内的方法和沉默肺TGF-β1基因对新生小鼠晚期肺泡发育的影响。方法:清洁级1日龄新生昆明小鼠75只,随机分为干扰组,空白对照组和阴性对照组3组,每组25只,于生后第3天（Postnataldays 3,以P3表示,下同）经鼻内吸入法将构建好的TGF-β1-shRNA慢病毒载体导入干扰组新生小鼠肺内,将阴性对照慢病毒载体同法导入阴性对照组小鼠肺内,空白对照组不予干预。各组按小鼠生后4,7,14,21,28天分别杀取5只小鼠取肺组织。左肺制作冰冻切片于激光共聚焦显微镜下观察肺组织红色荧光蛋白RFP的表达情况。左肺制作石蜡切片于光镜下观察肺组织形态学变化,计算肺泡平均截距（Mean linear intercept,MLI）代表肺泡腔内径大小,计算放射状肺泡计数（Radical alveolar counts,RAC）表示肺泡数目。取右肺采用逆转录聚合酶链反应（Reverse transcription-polymerase chain reaction,RT-PCR）法检测肺组织中TGFβ-1 mRNA表达水平。以Western Blot法检测肺组织中TGF-β1蛋白表达水平。数据统计用SPSS13.0软件行析因方差分析,多重比较用SNK法。结果:肺组织冰冻切片观察,在P4～P28各观察时间点均可见肺组织红色荧光表达;组织形态学观察,在P4各组肺形态无明显差别。干扰组P4 MLI和RAC与其他两组相比差异无统计学意义（P＞0.05）。与其他两组比较,干扰组P7～P28各时间点MLI明显增大（P＜0.05）,而RAC则明显减少（P＜0.05）,随时间呈进行性进展（分别F=68.898,P=0.000;F=87.520,P=0.000）。干扰组TGF-β1 mRNA表达水平在各时间点均明显低于空白对照组和阴性对照组（P＜0.05）;TGF-β1蛋白表达水平在三组相比,P4时各组差异无统计学意义（P＞0.05）,而P7～P28各时间点干扰组均明显低于其他两组（P＜0.05）;在各个时间点,空白对照组和阴性对照组相比上述各指标差异无统计学意义（P＞0.05）。结论:活体内鼻内吸入法可有效将慢病毒载体导入肺内,沉默新生小鼠肺组织TGF-β1表达可导致新生小鼠晚期肺泡发育过程阻滞。第三章小鼠支气管肺发育不良动物模型的建立及其肺组织TGF-β1、Smad 4的相关变化目的:以高体积分数氧吸入的方法制作小鼠支气管肺发育不良模型,研究高氧新生小鼠肺组织结构和TGF-β1、Smad4的变化情况。方法:清洁级1日龄新生昆明小鼠50只,于生后3天（Postnatal days 3,以P3表示,下同）随机分为高氧组和对照组两组,每组各25只。P4～P14将高氧组小鼠连同授乳母鼠一起置于自制氧箱内饲养。维持氧箱内氧气体积分数为600ml/L,以钠石灰吸附CO₂,使其体积分数低于5ml/L。供氧10天后恢复为空气条件饲养。空气组仅置空气条件下饲养;按P4,7,14,21,28每时间点各组分别杀取5只小鼠取肺组织,左肺制作石蜡切片,以HE染色法观察肺组织结构变化,计算肺泡平均截距（Mean linear intercept,MLI）代表肺泡腔内径大小,计数放射状肺泡计数（Radical alveolar counts,RAC）表示肺泡数目;制作超薄切片,透射电镜观察肺组织超微结构变化;以兔抗小鼠TGF-β1多克隆抗体为一抗,SABC免疫组织化学法观察TGF-β1在肺组织的表达情况;取右肺以RT-PCR法检测肺组织中TGFβ-1和Smad4 mRNA表达水平。数据统计用SPSS13.0行两独立样本t检验。结果:对照组生后4天可见新生小鼠肺泡结构不规则,肺泡数目较少,肺泡间隔较厚。P7开始对照组肺泡形态逐渐规整,间隔变薄,肺泡腔可见较多冠状突起,肺泡数目增加。P14肺泡发育大小已较均一。P21～P28肺泡发育逐渐成熟,肺泡数目多,肺泡腔较小,肺泡间隔薄。P4高氧组与对照组肺形态无明显差别,P7则可见高氧组肺泡腔和肺泡间隔炎性细胞渗出,肺泡间隔较厚,肺泡膈冠状突起较少,肺泡腔轻度扩大,P14～P21时肺泡腔进一步扩大并有融合,MLI明显增大,而RAC则明显减少,间质增生伴有程度不等的纤维化。至P28可见肺泡结构严重紊乱。在P7～P28,MLI和RAC两组相比均有统计学意义（P＜0.05）;电镜观察高氧组肺泡Ⅱ型上皮细胞肿胀,电子密度降低。胞质内板层小体较少或消失,结构松散,有排空现象。线粒体肿胀,体积增大。微绒毛脱落,排列紊乱。毛细血管内皮细胞肿胀明显;免疫组化显示对照组小鼠肺在P4～P28均有TGF-β1抗原阳性表达,主要表达部位为支气管上皮、血管内皮和肺间质。实验组在P7后表达较高氧组增强（P＜0.05）,肺泡上皮细胞也见明显阳性,特别是间质表达明显增强;两组P4时TGF-β1和Smad4 mRNA水平差异无统计学意义（P＞0.05）。高氧组P7～P28肺TGF-β1和Smad4 mRNA均较对照组明显升高（P＜0.05）。结论:成功建立新生小鼠高氧BPD模型。高氧吸入可导致肺组织TGF-β1和Smad4异常高表达,TGF-β1/Smad通路信号转导上调是支气管肺发育不良重要的病理机制之一。第四章慢病毒介导TGF-β1 siRNA对新生小鼠高体积分数氧吸入致肺损伤的保护作用目的探讨慢病毒介导的小分子干扰RNA技术抑制TGF-β1基因对高体积分数氧所致新生小鼠肺损伤是否具有保护作用。方法清洁级1日龄新生昆明小鼠100只,随机分为高氧组,干扰组,阴性干扰组和空气组4组,每组各25只,于生后3天（Postnatal days 3,以P3表示,下同）以鼻内吸入法将构建好的TGF-β1shRNA慢病毒载体及阴性对照载体分别导入干扰组和阴性干扰组小鼠肺内。P4～P14天将高氧组、干扰组和阴性干扰组置于氧箱内饲养。维持氧箱内氧体积分数为600ml/L,CO₂体积分数低于5ml/L。空气组不予任何干预;按小鼠生后4,7,14,21,28天每时间点各组分别杀取5只动物,左肺制作石蜡切片,HE染色观察肺组织形态学变化,计算肺泡平均截距（Mean linear intercept,MLI）代表肺泡腔内径大小,计算放射状肺泡计数（Radical alveolar counts,RAC）表示肺泡数目;以SP-C和AQP5单克隆抗体为一抗,SABC免疫组织化学法检测肺组织SP-C和AQP5表达情况;左肺制作冰冻切片,以FITC标记的荆豆凝集素（Ulex europaeus agglutinin 1,UEA-1）孵育后激光共聚焦显微镜观察绿色荧光表达情况;取右肺采用逆转录聚合酶链反应（Reverse transcription-polymerasechain reaction,RT-PCR）法检测肺组织中TGF-β1和ColleganⅠmRNA表达水平;以Western Blot法检测肺组织中TGF-β1蛋白表达水平。采集的数据用SPSS13.0统计软件行析因方差分析,多重比较用SNK法。结果P7后各时间点,干扰组MLI低于高氧组（P＜0.05）,高于空气组（P＜0.05）,而RAC则高于高氧组（P＜0.05）,低于空气组（P＜0.05）;免疫组织化学检测显示空气组AQP-5表达在P4～P28逐渐增强（F=614.440,P=0.000）,高氧组在P7高于空气组,后续时间点均较之明显降低（P＜0.05）。干扰组AQP-5表达在P14-28高于高氧组（P＜0.05）但低于空气组（P＜0.05）;空气组SP-C在P4～P14表达逐渐增强,到P14有最强表达,P21～P28表达下降（F=290.092,P=0.000）。高氧组SP-C表达在P7低于空气组,而在P14～P21均较之增强（P＜0.05）。干扰组SP-C表达在P14～P21较高氧组为低（P＜0.05）,但高于空气组（P＜0.05）。在P28空气组低于其他三组（P＜0.05）,其余各组无显著差别（P＞0.05）;冰冻切片观察UEA-1与肺微血管内皮细胞结合后FITC的表达情况,可见P14和P28各组FITC在肺微血管均有表达,空气组沿支气管和肺泡壁均见表达。高氧组和阴性干扰组则荧光表达增强,排列迂曲紊乱。干扰组较之高氧组和阴性干扰组荧光表达减弱,荧光分布与肺泡结构相一致;collagenⅠmRNA表达水平各组在P4～P7相比差异无统计学意义（P＞0.05）,干扰组P14后各时间点较高氧组和阴性干扰组为低（P＜0.05）,但高于空气组（P＜0.05）;干扰组P4 TGF-β1 mRNA水平明显低于其他各组（P＜0.05）。P7～P28干扰组低于高氧组和阴性对照组（P＜0.05）,但高于空气组（P＜0.05）;P4各组TGF-β1蛋白表达水平无差异（P＞0.05）。干扰组在P7后各时间点均低于高氧组（P＜0.05）,但高于空气组（P＜0.05）。高氧组和阴性对照组相比,上述指标在各时间点差异均无统计学意义（P＞0.05）。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有保护作用。抑制TGF-β1的异常高表达从而调节AECⅡ向AECⅠ的正常分化,促进细胞外基质正常降解,减轻肺微血管异常增殖并改善血管装配是其可能的保护机制。更多还原

【Abstract】 Bronchopulmonary dysplasia（BPD） is an important cause of premature infants morbidity and mortality.Patients who survive with BPD often have obstructive airway disease,pulmonary hypertension,and delay of growth and development. Despite recent advances in the care of premature neonates,the morbidity of BPD didn’t decline but even raised.There is no essential therapy for BPD at present which urge us to research pathogenesis of BPD and find effective treatments for it. Traditionally,Oxygen toxicity is one of important causes of BPD although the exact pathogenesis is not very clear.Previous studies have reported increased levels of transforming growth factor-β1（TGF-β1） in bronchoalveolar lavage fluid or lung tissues of BPD infants.However,there are fewer comprehensive studies about the role of TGF-β1 on BPD.Lentiviral vectors are capable of transducing a wide variety of dividing and nondividing cells,integrate stably into the host genome,and result in long-term expression of the transgene.And small interfering RNA（siRNA） technique is characterized by its specific silencing for taget gene.We constructed lentiviral vector expressing siRNA to explore whether inhibiting abnomal increased TGF-β1 in vivo does prevent lung from injury induced by hyperoxia in neonatal mice. Charpter one Construction and identification of lentiviral vector expressing siRNA of TGF-β1Objective:To construct the mouse TGF-β1-shRNA lentiviral vector and identify the knock-down efficiency of the TGF-β1 siRNA in alveolar epithelial cellsⅡ（AECⅡ）.Methods:The shRNA sequence of TGF-β1 and negative control sequence were designed respectively.The gene homology of sequences were confirmed by BLAST sequence alignment in PubMed.Annealing of sequences was processed in vitro to form double-stranded oligonucleotides.Ligation reaction was set up between annealed fragments and pRNAT-u6.2 vectors which digested by the BamH1/Xhol.The products of ligation were transformed into DH5αcompetent cells.The recombinant clones were validated through sequence analysis when the plasmids were extracted by a small amount of extraction method.The positive plasmids were digested by Xhol and Nhel,and 7010bp gene band was recycled from gel.PKG-RFP vector was digested by Xhol and Nhel,and 1262bp RFP DNA fragments were recycled and purified.Recombinant vectors containing the 7010bp and 1262bp DNA fragments were ligated by T4 DNA ligation reaction and transformed into DH5αcells.Plasmid DNA from the positive colonies can be screened by digestion with BamH1 restriction enzyme and a 600bp band would be seen from the positive lentiviral vector.Three lentiviral plasmids from Lentiviral packaging system and the positive recombinant vector were mixed together and plasmid mix were transfected into 293T cells to package the virus.Virus preparation was titrated by gradient dilution method using G418 of drug screening from collected virus supernatant.The negative control vector was constructed by the same way.For identification of down-regulation efficiency of the TGF-β1 siRNA in vitro,the ACEⅡwere randomly divided into 3 groups including interference group,negative control group and blank control group,for each set of five dishes.The packaged virus containing interference vector and negative vector were transfected into the plates of interference group and negative control group by polybrene method respectively,and the virus-free medium was added to the plate of blank control group.The down-regulation efficiency of TGF-β1 siRNA was verified to test the TGF-β1 protein expression by western blot after 96h transfection.Using the TGF-β1 polyclonal antibody as the first antibody,and FITC labeled goat anti-rabbit anti-IgG as the second antibody,the expression of TGF-β1 was observed by indirect immunofluorescence method on the AECⅡcells slices from 3 groups.Data were analyzed witt One-Way ANOVA and SNK method using SPSS13.0 software.Results:Sequencing analysis showed that,TGF-β1 shRNA silencing cassettes were inserted into pRNAT-u6.2 vector successfully.This recombinant plasmid was used to recombine with RFP fragments,and the positive Clones were identified by the procedures including transformation,screening of ampicillin-resistant and digestion of recombinant plasmid.The results of titration of virus preparation by gradient dilution method using G418 of drug screening showed that virus titer was 5.23×10⁸TU/ml.Western blot results showed that TGF-β1 protein expression of interference group was significantly lower than that of negative control group（P＜0.05） and blank control group（P＜0.05）,and down-regulation efficiency of lentiviral vector was 73.6%.Indirect immunofluorecent indicated that negative control group and blank control group had a strong fluorescent expression, and the expression of green fluorescent of interfence group was obviously weakening.Conclusion:Lentivial Vector of pU6.2-TGF-RFP-Lenti expressing TGF-β1 shRNA for mouse has been constructed successfully and the results showed it can knock down the AECⅡTGF-β1 protein expression effectively.Charpter two Influence of knock-down TGF-β1 gene on development of alveolar in neonatal miceObjective:To explore the method importing the lentiviral vector that expresses TGF-β1 siRNA into the lungs of neonatal mice and its impacts on alveolar development.Methods:75 1-day-old Kunming newborn mice were randomly divided into interference group,negative control group and blank control group,and 25 for each group.TGF-β1 shRNA lentivirus vector and control vector was administered intranasally to lungs of newborn mice in interference group and negative control group at postnatal days 3（Postnatal days 3,to express P3,the same below） respectively.No interference treatment was processed in blank control group. Lung samples of 5 mice from each group were excised at P 4,7,14,21,28 respectively for detecting the expression of red fluorescent protein on frozen sections using Laser scanning confocal microscope.Histomorphology analysis of lung tissues on paraffin sections was processed to check the mean linear intercept（MLI）of alveolar and the radical alveolar counts（RAC）.The expression of TGF-β1 mRNA and TGF-β1 protein in lung tissues from each group mice were determinated by RT-PCR and Western blot. Data were analyzed with univariate of general linear model and SNK of SPSS13.0 Software.Results:The expression of red fluorescent protein in lung tissue could be observed at P4～P28.The MLI and RAC in interference group were no significant difference compared with other two groups at P4（P＞0.05）.Compared with the other two groups,MLI of mice in interference group was increased while the RAC was reduced significantly at PT～P28（P＞0.05）.The expression of TGF-β1 mRNA in interference groups at all time point were lower significantly than those of blank control group and negative control group（P＜0.05）.The expression of TGF-β1 protein in interference groups were lower significantly than those of ohther two groups at all time point（P＜0.05） except P4.All results showed no significant difference between the blank control group andnegative control group at all time-point（P＞0.05）.Conclusion:The Intranasal administration is an efficacious method to import the lentiviral vector into the lungs of neonatal mice.Knock-down the expression of TGF-β1 gene by lentivirus vector-mediated small interfering RNA may lead to the developmental arrest of alveoli in neonatal mice.Charpter three The expression changes of TGF-β1 and Smad 4 in lung tissues of bronchopulmonary dysplasia miceObjective:Construction of mouse model of bronchopulmonary dysplasia induced by Oxygen exposoure and investigation changes of TGF-β1 and Smad 4 in lung tissues.Methods:50 newborn KM mice were randomly divided into hyperoxia group and control group.Each group contained 25 mice.Mice in hyperoxia group were exposed to 600mL/L oxygen from postnatal days 4 to days 14,and then restored to room air condition.The volume fraction of CO₂ was limited within 5ml/L.Mice in control group were exposed to room air condition.5 mice per group were randomly sacrificed and lung tissues were excised at postnatal days 4,7,14,21,28 respectively.Left lobes were processed for paraffin embedding and sectioning.Mean linear intercept（MLI） and radical alveolar counts（RAC） were counted.Ultrathin sections were made for observing ultrastructure of lung tissues by transmission electron microscope.SABC immunohistochemistry method was used to detect TGF-β1 in lung tissues.Right lobes were excised and frozen for detecting expression of TGF-β1 and Smad4 mRNA by Reverse transcription-Polymerase chain reaction.Data were analyzed with Independent-Sample T Test method using SPSS13.0 software.Results:Alveolar structure was irregular with thick alveolar wall and less alveoli number in every group at postnatal days 4.Mice in control group presented lung development from postnatal days 7 to days 28 characterized by increasing alveolar number along with more secondary septal crests formation.The thickness of alveolar wall and alveolar size decreased persistently with age.However,mice in hyeroxia group appeared impaired alveolar development characterized by a simplification of acinar structure, with a decreased number of alveoli and greatly enlarged terminal airways after postnatal days 7.Lung fibrosis was evidenced by increased thickness of interstitial after postnatal days 14.Some alveolar structure were ruined and fused after postnatal days 14.MLI of alveolar increased and RAC decreased in hyperoxia group compared with air group at postnatal days 7,14,21,28（P＜0.05）.In hyperoxia group, ultrastructure of lungs tissues showed that alveolar typeⅡepithelial cell were swollen along with the electron density decreased.Lamellar bodies,with a loose structure,reduced even dispeared.Swollen Mitochondria were also viewed. Microvilli exfoliated and arranged in disorder.Obvious swollen of capillary endothelial cells was observed.TGF-β1-staining mainly located at bronchial epithelial,vascular endothelial and interstitial of lungs at P4～P28 in control group.However,alveolar epithelial cells also were stained in hyperoxia group after P7. Compared with control group,the expression of TGF-β1-staining of lung tissues increased in hyperoxia group at postnatal days 7,14,21,28.There were no difference between two groups on expression of TGF-β1 and Smad 4 mRNA at P4.The expression of TGF-β1 and Smad 4 mRNA were higher significantly in hyperoxia mice than those of control mice at postnatal days 7,14,21,28.Conclusion:The mouse animal model for bronchopulmonary dysplasia was constructed successfully.High expressions of TGF-β1 and Smad 4 in lung tissues of newborn mice induced by hyperoxia suggest a key role of enhanced TGF-β1/Smad signaling in pathogenesis of bronchopulmonary dysplasia.Charpter four The protection against hyperoxia-induced lung injury in newborn mice by lentiviral vector-mediated TGF-β1 siRNA in vivoObjective:To explore whether down-regulating high levels of TGF-β1 by lentivirus vector-mediated small interfering RNA（siRNA） should protect against hyperoxia-induced lung injury in neonatal mice.Methods:100 newborn KM mice were randomly divided into hyperoxia group,interference group,negative control group and air group.Each group contained 25 mice.Lentiviral vectors containing TGF-β1 shRNA or nagetive control sequences were administered intranasally to lungs of mice in interference group and negative control group respectively at postnatal days 3.Mice in hyperoxia group,interference group,and negative control group were exposed to 600mL/L oxygen from postnatal days 4 to days 14 before restoring to room air condition.The volume fraction of CO₂ was limited within 5ml/L.Mice in air group were exposed to room air.5 mice per group were randomly sacrificed and lung tissues were excised at postnatal days 4,7,14,21,28 respectively. Left lobes were processed for paraffin embedding and sectioning.Mean linear intercept（MLI） and radical alveolar counts（RAC） were analyzed.SABC immunohistochemistry method was used to detect SP-C and AQP5 in lung tissues. Frozen sections were processed and incubated with FITC-UEA-1 for detecting microvascular proliferation and assembly by laser confocal microscope.Right lobes were excised and frozen for detecting expression of TGF-β1 and collagenⅠmRNA and TGF-β1 protein by Reverse transcription-Polymerase chain reaction and Western blot methods respectively.Data were analyzed with univariate of general linear model and SNK of SPSS13.0 software.Results:The MLI of alveolar in interference group was smaller than that of hyperoxia group but larger than that of air group at postnatal days 7,14,21,28（P＜0.05）.While the RAC was higher than that of hyperoxia group but lower than that of in air group at postnatal days 7,14,21,28（P＜0.05）;The expression of AQP-5 increased gradually from postnatal days 4 to days 28 in air group（F=614.440,P=0.000）.In hyperoxia group the AQP-5 expression were higher than that of air group at postnatal days 7 but lower than that of air group after that （P＜0.05） Interference group showed higher level of AQP-5 from P14～P28 compared with hyperoxia group but lower level compared with air group（P＜0.05）; The expression of SP-C increased gradually from postnatal days 4 in air group untill reaching peak value at postnatal days 14,then decreased and sustained a stable level after that（F=290.029,P=0.000）.SP-C staining in hyperoxia group was lower significantly than that of air group at postnatal days 7（P＜0.05） but higher than that of air group at postnatal days 14～21.The expression of SP-C in interference group was lower compared with hyperoxia group but higher compared with air group at postnatal days 14～21（P＜0.05）;Lectin-binding test showed the pattern of fluorescence was anfractuous and fluorescence intensity was enhanced in hyperoxia group and negative control group.However,fluorescence of FITC in interference group was consistent with alveolar structure and relatively weak;There were no difference among four groups on collagenⅠmRNA expression at postnatal days 4～7（P＞0.05）. At postnatal days 14～28,the expression of collagenⅠmRNA in lungs of interference group were lower significantly than those of hyperoxia group and negative control group（P＜0.05） but higher than those of air group （P＜0.05）;Compared with other three groups,the expression of TGF-β1 mRNA in lungs decreased significantly in interference group at postnatal days 4（P＜0.05）.At postnatal days 7,14,21,28,the expression of TGF-β1 mRNA and TGF-β1 protein in lungs of interference group were lower significantly than those of hyperoxia group and negative control group（P＜0.05） but higher than those of air group（P＜0.05）.The above indexes showed no significant difference between hyperoxia group and negative control group at every time-point.Conclusion:knock-down TGF-β1 gene by lentivirus vector-mediated small interfering RNA may protect against hyperoxia-induced lung injury in neonatal mice.Recovery of normal differentiation from AECⅡto AECⅠ,promotion of normal degradation of extracellular matrix and improvement of proliferation and assembly of pulmonary microvascular via down-regulating excessive levels of TGF-β1 may be important mechanisms of this protection.更多还原