环状RNA在小鼠肺发育中的连续表达及功能预测

目的 探讨环状RNA（circRNA）circ4:150439343|150477468和circ15:73330849|73343359在小鼠肺发育中的连续表达及潜在功能。方法 根据肺发育分期,取孕16.5 d（E16.5 d）、孕18.5 d（E18.5 d）及生后2 d（P2 d）胎鼠及新生小鼠的肺组织,利用苏木精-伊红染色观察肺组织形态学;利用qRT-PCR技术检测晚期肺发育过程中circ4:150439343|150477468和circ15:73330849|73343359的表达情况;应用miRanda和TargetScan软件预测circRNA的靶向miRNA,然后对靶基因进行GO和KEGG分析并预测相应circRNA功能。结果 在E16.5 d小鼠肺组织切片中观察到Ⅱ型肺泡上皮细胞,且逐渐增多,P2 d时肺泡迅速扩张,间质变薄,肺泡结构逐渐成熟;qRT-PCR结果显示circ4:150439343|150477468相对表达量随时间推移持续上调（P < 0.05）,circ15:73330849|73343359的相对表达量先下调再上调（P < 0.05）;宿主基因功能预测显示circRNA可参与Notch、PI3K-Akt和NF-κB等信号通路。结论 circ4:150439343|150477468和circ15:73330849|73343359可通过Notch等信号通路参与肺发育的调控。     

环状RNA在小鼠肺发育中的连续表达及功能预测

目的 探讨环状RNA（circRNA）circ4:150439343|150477468和circ15:73330849|73343359在小鼠肺发育中的连续表达及潜在功能。方法 根据肺发育分期,取孕16.5 d（E16.5 d）、孕18.5 d（E18.5 d）及生后2 d（P2 d）胎鼠及新生小鼠的肺组织,利用苏木精-伊红染色观察肺组织形态学;利用qRT-PCR技术检测晚期肺发育过程中circ4:150439343|150477468和circ15:73330849|73343359的表达情况;应用miRanda和TargetScan软件预测circRNA的靶向miRNA,然后对靶基因进行GO和KEGG分析并预测相应circRNA功能。结果 在E16.5 d小鼠肺组织切片中观察到Ⅱ型肺泡上皮细胞,且逐渐增多,P2 d时肺泡迅速扩张,间质变薄,肺泡结构逐渐成熟;qRT-PCR结果显示circ4:150439343|150477468相对表达量随时间推移持续上调（P < 0.05）,circ15:73330849|73343359的相对表达量先下调再上调（P < 0.05）;宿主基因功能预测显示circRNA可参与Notch、PI3K-Akt和NF-κB等信号通路。结论 circ4:150439343|150477468和circ15:73330849|73343359可通过Notch等信号通路参与肺发育的调控。     

血管内皮生长因子和内皮抑素在大鼠肺的表达及其与肺发育的关系

目的探讨大鼠肺发育过程中血管内皮生长因子(VEGF)和内皮抑素(ES)的表达及其与肺发育的关系。方法选取健康SD大鼠,雌雄按3∶1合笼交配,孕鼠随机分为8组,分别于胚胎18、20、21天,生后1、4、7、14、21天处死孕鼠或幼鼠取胎鼠或幼鼠肺标本,每个时间点取8只大鼠的肺组织,一部分进行Western blot检测VEGF及ES蛋白表达,另一部分行病理切片HE染色观察肺组织形态。结果随胎龄及生后日龄增长,胎鼠/幼鼠肺泡数目明显增多,肺泡壁变薄,间质减少,肺泡腔扩大,肺发育逐渐成熟。胚胎18、20、21天,生后1、4、7、14、21天胎鼠/幼鼠肺组织VEGF蛋白相对表达量分别为(0.85±0.04)、(0.65±0.15)、(0.42±0.08)、(0.79±0.11)、(1.05±0.06)、(0.87±0.03)、(1.31±0.14)、(1.70±0.05),ES蛋白相对表达量分别为(0.47±0.04)、(0.35±0.01)、(0.74±0.11)、(0.36±0.03)、(0.52±0.06)、(0.30±0.09)、(0.22±0.09)、(0.17±0.04),新生鼠出生前(胚胎21天)VEGF蛋白表达量较胚胎18、20天明显降低,至出生后14天再次出现增长高峰,生后21天达最高水平。新生鼠出生前(胚胎21天)ES蛋白表达量较胚胎18、20天及生后1、4天均明显增高,而后维持在较低水平。VEGF与ES蛋白表达水平呈负相关(r=-0.752,P=0.032)。结论 VEGF和ES参与肺发育过程,其表达变化对肺发育起着重要作用。     

CyclinD1和p21CIP1在大鼠肺发育过程中的动态表达

目的：细胞周期素D1（CyclinD1）、p21CIP1是主要的肺细胞增殖调控蛋白,参与肺发育、肺损伤修复过程。本研究观察大鼠肺发育过程中小管期、囊泡期、肺泡期等不同阶段CyclinD1和p21CIP1的表达特征。方法：分别于孕20 d、21 d、生后0 d、3 d、7 d、14 d、21 d取胎鼠或新生鼠肺组织标本（n=6）,用免疫组织化学技术检测CyclinD1和p21CIP1在大鼠肺发育各期定位表达,免疫印迹半定量检测大鼠肺发育过程中CyclinD1和p21CIP1蛋白的表达。结果：在大鼠肺发育各期中,小管期CyclinD1蛋白表达最强,肺泡期CyclinD1蛋白表达最弱。而p21CIP1蛋白表达在小管期最弱,肺泡期最强。 在小管期CyclinD1阳性表达主要定位于上皮细胞,p21CIP1表达阴性。囊泡期CyclinD1表达强度明显减弱,CyclinD1 和p21CIP1阳性表达主要定位于上皮细胞和间质细胞。肺泡期p21CIP1表达最强,CyclinD1阳性表达主要定位于间质细胞,p21CIP1阳性表达主要定位于上皮细胞。结论：大鼠肺发育过程中CyclinD1和p21CIP1在各期表达部位及数量有差异,在小管期, CyclinD1表达最强,细胞增殖活动明显较囊泡期和肺泡期旺盛,肺泡期p21CIP1表达最强与肺泡分隔及成熟有关。     

胰岛素样生长因子结合蛋白2在新生鼠肺发育中的作用

目的探索胰岛素样生长因子结合蛋白(IGFBP)2在新生鼠肺发育中的作用。方法SD孕鼠80只分为4组,即A:对照组;B:地塞米松(Dex)1组;C:Dex2组;D:维A酸(RA)组。A、B、D组分别于孕18～20d皮下注射生理盐水、Dex,腹腔注射RA;C组于生后1～3d皮下注射Dex。各组分别于孕18、20、21d(C组孕期不取)、生后1、3、5、7、10、14、21d取肺标本作形态学检查、免疫组织化学、Westernblot及RT PCR检测。结果1.肺组织形态学:B、C组早期肺泡发育提前,数目多,壁薄,晚期则明显落后于A、D组。2.IGFBP2肺表达:A组中IGFBP2主要在胎肺组织中表达,孕18d左右表达最强,随后表达渐减弱。B、C组表达趋势同A组,但生后各时间点均较A组增强;D组生后各时间点均较A组减弱。3.Westernblot:IGFBP2多肽表达强度各组孕18d表达最强,随后渐降低;B、C组生后各时间点表达强度明显高于A组(P均<0.01);D组各时间点浓度均明显低于A组(P均<0.01)。4.RT PCR:IGFBP2mRNA表达的强度变化规律与其肽浓度变化相似。结论IGFBP2在肺发育过程起重要作用,其浓度过高则影响肺发育。     

Sprague-Dawley大鼠肺发育中miR-431的连续表达研究

目的 探讨miR-431在肺发育形态学中的潜在作用。方法 根据肺发育分期,选择SpragueDawley大鼠孕16 d（E16）、孕19 d（E19）、孕21 d（E21）、生后1 d（P1）、生后3 d（P3）、生后7 d（P7）、生后14 d（P14）及生后10周（P10周）成年大鼠的肺组织作为观察点,利用苏木精-伊红染色（HE）及透射电镜方法进行肺组织形态学观察;利用荧光原位杂交和实时荧光定量PCR技术检测肺发育关键时期（E19、E21及P3）肺组织中miR-431的表达情况。结果 E19组胎肺组织发现关键事件板层小体及Ⅱ型肺泡上皮细胞的出现;随胎龄增加板层小体数量增加,并出现聚集与排出;生后肺泡形成迅速发生,间质变薄,微血管系统逐渐成熟。荧光原位杂交及实时荧光定量PCR结果均显示随着胎龄的增加,miR-431的表达量逐渐降低（P < 0.05）。结论 获得系统而连续的肺发育形态学资料。miR-431在肺发育过程中可能起重要的负向调控作用,为后续进一步研究肺发育及相关性疾病的机制提供基础和方向。     

盐酸氨溴索、地塞米松产前用药对大鼠胎肺形态发育影响的对比

目的比较产前3d给盐酸氨溴索、地塞米松及单次给地塞米松对大鼠胎肺形态发育的影响。方法孕鼠12只随机分为9g／L盐水对照组和产前盐酸氨溴索3d治疗组、地塞米松3d及1d治疗组4组，每组3只。孕d19每只孕鼠取6只胎鼠肺组织，通过光镜观察及电镜技术比较2种药物及不同疗程对胎肺形态发育的影响。结果1．光镜下3个治疗组每视野肺泡计数、平均肺泡表面积均高于对照组（Pa〈0．01），平均肺泡间隔厚度均低于对照组（Pa〈0．01）。2．光镜下地塞米松3d治疗组每视野肺泡计数、平均肺泡表面积均高于地塞米松1d治疗组，平均肺泡间隔厚度低于地塞米松1d治疗组（Pa〈0．01）。3．光镜下地塞米松3d及1d治疗组每视野肺泡计数、平均肺泡表面积均高于盐酸氨溴索治疗组，平均肺泡间隔厚度低于盐酸氨溴索治疗组（Pa〈0．01）。4．透射电镜观察：治疗组Ⅱ型肺泡上皮内多见板层小体，尤以地塞米松3d治疗组更为多见，且染色深、致密，线粒体等细胞器多；对照组难见板层小体，少见细胞器。结论产前应用地塞米松、盐酸氨溴索均能显著促进胎肺发育；多次地塞米松优于单次地塞米松治疗；多次及单次地塞米松疗效均优于盐酸氨溴索治疗。     

Notch2、Notch4在新生早产大鼠高氧肺损伤中的表达

目的探讨Notch2、Notch4在早产大鼠高氧肺损伤发生发展中的作用。方法将生后1d内早产SD大鼠随机分为高体积分数氧暴露组和空气组。于生后1、7、14、21d留取肺组织标本,免疫组织化学法检测Notch2、Notch4的表达,RT-PCR检测Notch2、Notch4 mRNA的表达。结果在肺发育不同时期,Notch2、Notch4表达具有各自不同的规律,吸氧体积分数为85%氧暴露不同程度改变了它们的表达。结论在吸氧体积分数为85%的氧气长期暴露,Notch2、Notch4异常表达,可能是早产大鼠肺损伤的发生机制之一。     

盐酸氨溴索和倍他米松对大鼠胎肺形态发育影响的对比研究

目的 探讨产前注射盐酸氨溴索和倍他米松对大鼠胎肺形态发育的影响.方法 12只孕鼠随机分成4组:盐酸氨溴索组、倍他米松1d组、3d组及对照组,每组3只大鼠.盐酸氨溴索组、倍他米松3d组从妊娠第16天起分别腹腔注射3d盐酸氨溴索100mg/(kg·d)、倍他米松O.2mg/(kg·d);倍他米松1d组在妊娠第16、17天注射生理盐水,第18天注射倍他米松0.2mg/(kg·d);对照组在同样时间连续3d注射生理盐水.在妊娠第19天将孕鼠处死后立即剖腹取仔鼠,每只孕鼠取6只胎鼠肺组织,通过光镜观察、图像分析及电镜技术比较2种药物、不同疗程对孕鼠的胎仔肺组织形态结构影响.结果 光镜下各治疗组与对照组相比肺泡间隔薄(P<0.001),呼吸膜周径及肺泡表面积均增大(P相似文献   

轻度维生素A缺乏对不同时期胎鼠肺发育的形态学影响

目的 观察轻度维生素A缺乏(MVAD)对不同时期胎鼠肺发育的形态学影响.方法 24只SD雌鼠随机分为MVAD组(n=15)和对照组(n=9).通过喂养缺乏维生素A(VA)饲料建立MVAD孕鼠模型,高效液相色谱法检测2组雌鼠血清VA水平和孕20 d胎鼠肝脏VA水平.取孕16 d、20 d、出生1 d MVAD组和对照组胎(乳)鼠肺组织,经HE染色,光镜下观察其肺形态学改变,比较MVAD组和对照组鼠血清VA、孕20 d肝VA水平及胎肺发育的形念学区别.应用SPSS 13.0软件进行统计学分析.结果 MVAD组雌鼠毛色干枯,少食少动,孕鼠体质量下降,部分流产不孕,平均孕胎数较对照组下降(P＜0.05);出生1 d MVAD组乳鼠体质世、身长、尾长值均较对照组小(Pa＜0.01).MVAD组雌鼠血清VA水平显著低于对照组(P＜0.01),孕20 d胎鼠肝脏VA水平MVAD组亦显著低于对照组(P＜0.01).HE染色显示MVAD组16 d胎鼠支气管圆形管腔较对照组分支样管腔幼稚;MVAD组20 d胎鼠较埘照组原始肺泡少,平均肺泡表面积较小,肺泡间隔较厚(P＜0.01);MVAD组出生1 d乳鼠表现为肺发育的不均一性,较多区域有肺泡过度膨胀,肺泡间隔较薄(P＜0.01),有离断;局部又有肺泡膨胀小全,间质充血,肺泡间隔增厚.结论 VA在肺发育中起重要作用,MVAD会导致胎鼠肺发育小全.     

Ontogeny of surfactant apoproteins in the rat

Content of the 26-38-kD surfactant apoprotein (SP-A) was determined in lung homogenates from fetal (17-21 d gestation), postnatal (1-28 d of life), and adult male and female rats by a double sandwich ELISA. Expression of mRNA for SP-A as well as the hydrophobic apoproteins, SP-B and SP-C, were also determined in lung homogenates from fetal and adult rats of both sexes by Northern blot analysis. SP-A was undetectable in fetal lungs on d 17 (day of birth = d 22) and barely detectable on d 18. On d 19 there was a 3- to 4-fold increase in SP-A content above d 18 levels. Between d 19 and 21 SP-A content significantly increased another 6- to 9-fold. SP-A content on the day of birth was not significantly different from that seen on gestational d 21. SP-A content decreased 35-40% between the day of birth and postnatal d 7. After the second postnatal week SP-A content gradually increased, reaching adult levels after d 28. No sex differences in SP-A content were observed during fetal or postnatal lung maturation. SP-A mRNA was first detected in fetal lungs on d 18 and increased in relative abundance until d 21, but remained below adult levels. Developmental changes in fetal lung SP-A content closely paralleled changes in fetal expression of SP-A mRNA. SP-B mRNA was also first detected on d 18, then increased in relative abundance to adult levels by d 20. SP-C mRNA was clearly detectable on d 17, then increased in relative abundance to adult levels by d 20-21. Unlike surfactant phospholipids, there are no apparent sex differences in the expression of any of the surfactant apoproteins during late gestation. The differences observed during fetal lung maturation in the time of onset and changes in relative abundance among the three apoprotein mRNA imply that their genes may be differentially regulated in the developing rat lung.     

Repetitive prenatal glucocorticoids increase lung endothelial nitric oxide synthase expression in ovine fetuses delivered at term

Antenatal administration of glucocorticoids has been shown to improve postnatal lung function after preterm birth in the ovine fetus. Mechanisms of steroid-induced lung maturation include increased surfactant production and altered parenchymal lung structure. Whether steroid treatment also affects lung vascular function is unclear. Because nitric oxide contributes to the fall in pulmonary vascular resistance at birth, we hypothesized that the improvement of postnatal lung function of preterm lambs after treatment with prenatal glucocorticoids may be in part caused by an increase in endothelial nitric oxide synthase (eNOS) activity. To determine whether glucocorticoid treatment increases lung eNOS expression, we measured eNOS protein content by Western blot analysis of distal lung homogenates and immunostaining of formalin-fixed lungs from ovine fetuses delivered at preterm and term gestation after prenatal administration of glucocorticoids. Treatment protocols were followed in which ewes were treated with intramuscular betamethasone (0.5 mg/kg) at single or multiple doses at weekly intervals, and fetuses were delivered at 125, 135, or 145 d gestation. All groups were compared with saline-treated controls. Western blot analysis of whole lung homogenates demonstrated a 4-fold increase in eNOS protein content in lambs treated with repetitive doses of glucocorticoids and delivery at term (145 d; p < 0.002). In addition, a small increase in lung eNOS protein content was seen in lambs treated with a single dose of betamethasone at 128 d gestation with delivery at 135 d gestation. In comparison with control animals, there were no differences in lung eNOS content from the remaining lambs treated with glucocorticoids when delivery occurred at preterm ages (125 and 135 d). Immunostaining showed eNOS predominantly in the vascular endothelium in all vessel sizes. Pattern of staining was not altered by treatment with antenatal glucocorticoids. We conclude that maternal treatment with glucocorticoids increases lung eNOS content after multiple doses and delivery at term gestation. We speculate that antenatal glucocorticoids may up-regulate eNOS but that the timing and duration of steroid administration appears to be critical to this response.     

半胱氨酸蛋白酶3和白细胞介素8在高氧暴露下早产大鼠肺组织中的动态表达及其意义

目的 探讨半胱氨酸蛋白酶-3(Caspase-3)及白细胞介素-8(IL-8)在早产大鼠高氧肺损伤中的动态表达及其意义.方法 孕21 d的SD早产大鼠生后第2天,随机分为空气组和高氧组(均n=40).高氧组大鼠予以85%高氧持续暴露,空气组大鼠置于空气中.于暴露1、4、7、14和21 d每组各处死8只大鼠,收集肺组织标本,苏木精-伊红染色观察肺组织病理形态学变化,双抗夹心ELISA法检测IL-8含量,免疫组化和Western blot检测Caspase-3表达.结果 高氧暴露后肺泡腔内可见有坏死脱落细胞、炎症细胞渗出增多、间质水肿,肺组织结构紊乱,肺泡形成明显滞后,肺泡结构简单化和囊泡化;与空气对照组比较,高氧暴露4、7和14 d肺组织Caspase-3和IL-8含量均明显增高(P＜0.01).结论 在高氧所致早产大鼠肺损伤中细胞凋亡和坏死共存,两者共同参与了早产大鼠高氧肺损伤的病理过程.     

Alveolar development in the human fetus and infant

The lungs from 29 infants aged from 29 weeks of gestation to 18 weeks postnatal age were studied using morphometric analysis; total DNA was estimated in 12 of these. Alveoli could first be counted and measured at 29 weeks gestation; with increasing age they became more mature in appearance as the walls elongated and thinned, and they gradually increased in diameter. Lung volume increased 4-fold between 29 weeks and term, and further doubled in the 4 months after birth. Lung volume, alveolar surface area and DNA all increased linearly with age and weight. Alveolar number showed a curvilinear increase with age and DNA, but a linear relationship to body weight. At birth the lungs had an average of 150 million alveoli, half of the expected adult number. There was a wide normal range. The surface area was between 3 and 5 m2 at birth, one twentieth of the adult value.     

Antenatal dexamethasone administration impairs normal postnatal lung growth in rats

Our purpose was to determine the influences of antenatal dexamethasone administration on neonatal lung development in rats. Dexamethasone (0.4 mg/kg maternal body weight per day) was administered i.p. on the 21st d (group 1) or on the 20th and 21st d (group 2) of gestation in Sprague Dawley rats with timed pregnancies. After natural deliveries, the lungs of the pups 1-60 d of age were removed and processed for morphometric analysis. In 60-d-old pups, groups 1 and 2 both showed a lower numerical density of alveoli and a larger mean alveolar radius than control pups. Antenatal administration of dexamethasone to rats impairs the normal postnatal lung growth. Some aspects of the use of antenatal glucocorticoid therapy in humans may require reconsideration.     

Effect of premature delivery on rat lung retinol (vitamin A) and retinyl ester stores

Low plasma retinol (vitamin A) in premature infants has been associated with bronchopulmonary dysplasia. Also, retinol (vitamin A) deficiency is characterized by loss of cilia and squamous metaplasia of the respiratory tract, similar to the histological lesions of bronchopulmonary dysplasia of the premature infant. Recent work showed that the fetal rat lung retinyl palmitate stores were high preterm, but fell abruptly between 21 days of gestation and postpartum. This report extends this observation by showing that lung retinyl palmitate also decreases after cesarean section delivery preterm. Lung retinol and retinyl palmitate in the lungs of 21-day gestation fetuses delivered by cesarean section and stimulated to breath were compared to their in utero littermates. In utero lungs contained 2.2 +/- 0.14 micrograms/g dry weight retinol and 14.9 +/- 0.8 micrograms/g dry weight retinyl palmitate. Lungs from littermates that had been removed from the uterus and breathed on their own for 4 h had lower retinol (1.6 +/- 0.08 micrograms/g) and retinyl palmitate (11.3 +/- 0.4 micrograms/g; p less than 0.01). Fetal lung contains retinyl palmitate stores that are readily mobilized with delivery at term or preterm.     

宫内注射脂多糖对围产期大鼠肺Toll样受体4信号转导通路的影响

目的 观察宫内注射脂多糖(LPS)对围产期大鼠肺内天然免疫相关的Toll样受体4(TLR4)信号转导通路的影响,探讨天然免疫在宫内感染中的免疫调节能力及对肺发育的影响.方法 将30只孕17d的SD大鼠随机分为LPS组和生理盐水对照组,LPS组宫内注射LPS 10μl(40μg/ml),对照组宫内注射等体积的灭菌生理盐水.分别留取胎龄18、20、22 d(E18、E20、E22)的胎鼠肺组织、胎盘组织标本以及生后1、3、7d(P1、P3、P7)新生鼠肺组织标本,HE染色观察病理改变,RT-PCR技术检测TLR4、髓样分化因子88(MyD88)和白介素1 β(IL-1β)mRNA表达,免疫组织化学技术检测肺组织TLR4、MyD88的表达分布情况.实验数据采用单因素方差分析和q检验进行统计学分析.结果 (1)LPS组孕鼠胎盘组织有大量中性粒细胞浸润,宫内感染模型建立成功;(2)在E18、E20和E22时,LPS组胎鼠肺组织无明显病理学改变,以后逐渐出现改变,于P7时可见肺泡数量减少,肺泡腔变大,间隔变薄,但未见明显结构紊乱;(3)LPS组TLR4、MyD88和IL-1β mRNA水平于E20和E22均高于对照组,差异有统计学意义(P<0.05),且均于E22表达达高峰,后缓慢下降;(4)免疫组织化学结果显示E18时两组肺组织内均未见明显TLR4和MyD88阳性染色,后均逐渐表达增加,且主要在细支气管和肺泡上皮细胞表达.结论 (1)宫内注射LPS可导致胎鼠和早产鼠肺组织TLR4、MyD88表达在一定范围内增加,后逐渐回复正常水平,同时肺组织的病理改变和炎症反应较为温和,推测在围产期胎肺天然免疫系统可以调节LPS诱导的炎症反应强度;(2)该实验在一定程度上证实宫内感染激活的信号转导通路是MyD88依赖性途径.