胎肺发育中相关活性物质的表达及其调控意义

目的 观察表皮生长因子受体(EGFR)、转化生长因子β(TGF-β)和甲状腺转录因子-1(TTF-1)在胎肺发育过程的表达，探讨它们的生物活性作用及相互之间的调控意义。方法 应用免疫组织化学方法，检测EGFR、TGF-β(β1、β2、β3)和TTF-1因子在小鼠胎肺组织中的表达，用TIGER图像分析系统，对TTF-1表达强度进行定量分析。结果 胎肺发育的早期，EGFR定位于近端支气管上皮，TTF-1表达于远端支气管上皮细胞核内；而TGF-β1、β2和β3的表达定位基本一致，呈区域性分布于近端支气管上皮。到发育的中、后期，EGFR和TTF-1因子定位于肺泡上皮细胞，呈强表达，TGB-β则主要存在于间质；图像分析结果显示：在胎肺发育过程，TTF-1阳性细胞的平均光密度(AA)呈逐渐增加，而体积(VOL)和体积积分光密度(VIA)却逐渐减小。结论 EGFR、TGR-β和TTF-1因子在胎肺不同发育阶段具有不同表达特征，表明可能发挥着不同的功能，它们对支气管树的构筑、呼吸道和肺泡上皮的成熟分化都有重要的调节作用。     

人胎肺发育过程中生存素表达及生物学意义

目的检测生存素(survivin)和caspase-3在人胎肺发育过程的表达特征,探讨两者在胎肺发育中的意义。方法采用16～35周人胎肺组织,用免疫组化SP法检测survivin和caspase-3的表达。结果在胎肺的假腺期和小管期,survivin主要表达于远端支气管上皮,在原始肺泡期,survivin阳性表达于原始肺泡上皮,到35周,近端支气管上皮细胞中出现散在的survivin阳性细胞。caspase-3在支气管上皮的表达,以小管末期最强,主要定位于近端支气管上皮、Ⅱ型肺泡细胞。结论 胎肺发育早期,survivin对支气管树形态构筑过程中上皮细胞的发育与分化具有重要的保护意义,caspase-3参与支气管树的重建。胎肺发育晚期,Survivin和caspase-3协同作用,调控着Ⅰ型和Ⅱ型肺泡细胞的成熟和分化。     

人胎肺Ⅱ型肺泡细胞中表面蛋白-B的表达及意义

目的检测肺表面蛋白-B(SP-B)及其调控因子甲状腺转录因子-1(TTF-1)在肺泡Ⅱ型细胞发育过程的表达特征,探讨两者对于人胎肺肺泡Ⅱ型细胞发育、分化和成熟的调控作用。方法取16-35周人胎肺组织,常规石蜡包埋切片,用免疫组化技术检测SP-B和TTF-1的表达特征。结果,TTF-1在胎肺16周开始表达,定位于上皮细胞核内,随支气管树的发育分化,远端位置的反应总是较近端者强。SP-B蛋白于18周开始表达,定位于肺泡Ⅱ型细胞胞浆内,阳性反应细胞于早、中期表达丰富;由呼吸道近端逐渐往远端迁移,且强度不断增强。发育末期至成肺中,TTF-1和SP-B阳性反应均在肺泡Ⅱ型细胞中稳定表达。结论TTF-1参与支气管树形态发生和肺泡的发育成熟,并调控肺泡Ⅱ型细胞中SP-B蛋白的分泌,起稳定肺泡直径的作用。因此,SP-B蛋白的分泌反映肺泡Ⅱ型细胞功能的成熟。     

胎肺中甲状腺转录因子-1的表达及其生物学作用

目的　检测甲状腺转录因子 1 (TTF 1 )在小鼠胎肺发育过程中的表达特征 ,探讨其对胎肺发育、分化和成熟的调控作用。方法　不同发育阶段昆明小鼠胎肺组织 ,用免疫组织化学技术观察TTF 1的表达特征 ,用Tiger图象分析系统进行定量分析。结果　小鼠胎肺 1 0d开始检测到TTF 1的表达。其主要表达于正在形成呼吸道的上皮细胞核内 ,且处于末端位置的反应总是明显较近端者强 ;出生后 ,TTF 1主要表达在Ⅱ型上皮细胞核内。在胎肺发育过程 ,TTF 1的AOD值逐渐增加 ,表明Ⅱ型肺泡上细胞TTF 1密度呈现增加趋势 ,与肺泡功能的不断分化成熟相适应。结论　TTF 1参与肺组织的发生 ,可能具有调控上皮细胞发育的重要功能 ;肺泡发育过程中 ,TTF 1在Ⅱ型细胞的丰富表达 ,提示可能与肺泡功能的成熟分化 ,适应出生后执行功能的需要密切相关。     

胎儿肺Ⅱ型肺泡细胞发育中相关活性物质的表达及其意义

目的探讨诸细胞因子对肺泡Ⅱ型细胞发育、分化和成熟的调控作用。方法12～35周胎儿肺组织共13例,常规石蜡包埋、切片,用免疫组织化学技术检测FGFR、Ras-p21蛋白、BMFL4、SP-B和TTF-1的表达特征。结果FGFR率先在发育早期近端支气管上皮表达。Ras蛋白于16周在近端和远端的支气管上皮有较弱表达,18周时与FGFR一样达到表达的高峰。随着FGFR表达的增强,BMF-4开始表达。发育后期,这三者均不表达。TTF-1因子自16周起定位于上皮细胞核内,远端支气管的反应总是较近端者强。SP-B蛋白在18周胎儿肺Ⅱ型肺泡细胞内出现,伴随着TTF-1的表达,其反应由呼吸道近端逐渐往远端迁移。发育末期至成肺中,TTF-1和SP-B阳性反应均在肺泡Ⅱ型细胞中稳定表达。结论FGF-10、Ras蛋白和BMP-4对Ⅱ型肺泡细胞的作用,是通过细胞外间质的途径促使Ⅱ型肺泡细胞正常增殖分化和达到形态结构的成熟;而TTF-1因子和SP-B的表达,则为Ⅱ型肺泡细胞在胎肺发育晚期已逐渐达到功能成熟的标志,与胎儿一出生即具有自主呼吸的功能相适应。     

胎儿发育中水通道蛋白的免疫组化观察

目的观察胎儿器官和组织发育过程中水通道蛋白(AQPs)的表达特征,初步探讨AQPs对胎儿发育进程的生物意义。方法14～38周胎儿共12例,取用肾脏、肺脏、唾液腺、甲状腺和胃等器官,常规固定、石蜡包埋和切片;用免疫组化S-P法,检测AQPs(AQP1、AQP2和AQP4)在胎儿不同胎龄器官组织中的表达。结果胎儿肾脏中AQPs定位于近端小管和集合管系统;胎肺中AQPs的表达随肺泡发育分化而迁移,始终定位于肺泡上皮;晚期胎肺中AQPs于肺泡及呼吸道上皮均有表达;胎16周起检出AQPs反应于唾液腺、胃腺和胰腺等消化腺,同时甲状腺中AQPs在滤泡上皮也有活跃表达。结论胎肾中AQPs的表达与肾的重吸收功能直接相关;胎肺内AQPs的表达反映肺泡上皮分化的轨迹,其调节水分的作用为肺泡发育提供空间;AQPs介导细胞内外水分的转运不但调节消化腺的分泌还参与调节甲状腺滤泡的激素合成和分泌过程。表明AQPs在胎儿发育过程,对各器官中水转运功能的成熟起重要作用。     

小鼠肺泡发育与肺泡上皮细胞分化的电镜观察

目的　观察小鼠胚胎及生后肺泡发育及肺泡上皮分化。方法　小鼠胚胎 14天至生后 14天肺组织 ,隔天取材 ,HE染色光镜观察及透射电镜观察。结果　胚胎 14～ 18天 ,小鼠肺的发育以支气管树分支和管壁结构的逐渐完善为主 ,终蕾上皮为柱状或立方状的未分化细胞。胚胎 19天 ,支气管远端形成许多内壁光滑的原始肺泡 ,其上皮分化出Ⅱ型肺泡细胞。生后 1～ 4天 ,肺泡上皮出现少量扁平的Ⅰ型肺泡细胞 ,但仍以Ⅱ型肺泡细胞为主。生后 5～ 14天 ,成熟肺泡形成 ,肺泡上皮以Ⅰ型肺泡细胞为主。结论　出生时 ,小鼠的肺发育只完成了其大体形态的发生 ,肺泡上皮以Ⅱ型肺泡细胞为主。成熟肺泡的形成、数量的增加及Ⅰ型肺泡细胞的大量出现持续到出生后。     

小鼠胎肺发育中肺泡上皮细胞的电镜观察

<正> 小鼠胚胎第12天至生后7天的肺组织,隔天取材,共15例,常规电镜标本制作,透射电镜观察,探讨胎肺发育过程中肺泡上皮细胞的超微结构变化.观察结果表明:胚胎第12～14天(相当于人胎肺的假腺期)肺内均为管状“肺泡”,由形态相同的柱形细胞围成,核椭圆形,核膜平整,有1-2个核仁.细胞内含高尔基氏体、线粒体、粗面内质网和糖原.细胞表面有微绒毛,细胞之间有连结复合体.胚胎第16～18天,胞质内出现大量的糖原颗粒.胚胎第19天(相当于人胎肺的原始肺泡期),肺泡上皮细胞多为立方形,出现Ⅱ型肺泡细胞,其特征为表现有微绒毛,胞质内糖原减少,出现椭圆形板层小体,长约1～2μm,外有界膜,内含平行排列的板层,肺泡腔内偶可见板层小体.同时,肺内支气管上皮分化出纤毛细胞,其胞质电子密度低,纤毛的基粒处有线粒体.新生 1～4天,肺泡上皮仍以Ⅱ型细胞为主,Ⅱ型细胞内板层小体及线粒体数目增多,线粒体基质较致密.开始出现少量扁形的I型肺泡细胞.致新生7天,肺泡上皮才分化为以I型细胞为主.结论:胚胎18～19天以前胎肺内以形成结构分化为主,胚胎19天是Ⅱ型肺泡细胞分化的关键时期,而Ⅰ而型     

EGFR与PDGFR-α在肺腺癌中的表达及意义

目的探讨血小板衍生生长因子受体（PDGFR-α）和表皮生长因子受体（EGFR）与肺腺癌发生发展的关系。方法免疫组化SP法检测PDGFR-α与EGFR在60例肺腺癌原发灶和24例正常支气管黏膜中的表达。结果 PDGFR-α主要在肺腺癌细胞质表达,少数在细胞核表达;EGFR主要表达于肿瘤细胞的细胞膜上,细胞浆中也有表达。二者均在肺腺癌细胞中过度表达（P〈0.05）。PDGFR-α的阳性表达率在肺腺癌高分化组明显高于低分化组（P〈0.05）,而EGFR在肺腺癌高分化组和低分化组中的表达相仿（P〉0.05）。结论 PDGFR-α的阳性表达与肺腺癌的分化程度呈正相关,EGFR的阳性表达与肺腺癌的分化程度无相关性。     

花背蟾蜍肺发生及转化生长因子-β的表达

目的分析花背蟾蜍肺发生的规律,观察转化生长因子-β(TGF-β)在肺发育过程中的表达,探讨其生物活性作用及调控意义,为动物学和发育生物学研究提供基础资料。方法利用生物显微技术和免疫组织化学方法,研究花背蟾蜍肺(218只)的组织发生,检测TGF-β(TGF-β1、TGF-2β、TGF-3β)在花背蟾蜍肺组织发生中的表达,并利用IPP专业图像分析软件对其表达强度进行定量分析。结果1.花背蟾蜍的肺发生开始在幼虫第22期,它起始于咽部尾侧,从26期开始分化出微小腔隙,到31期开始形成明显肺腔,33期开始出现原始肺泡,36期肺内出现明显的隔膜和肺泡,肺壁中已有较多毛细血管,39期肺已接近幼蟾肺,肺上皮由矮柱状或立方形变成扁平形。整个发育过程可分为4个阶段,即肺芽Ⅰ期、肺芽Ⅱ期、原始肺泡期和肺泡期。2.在蟾蜍发育早期,TGF-β(β1、β2、β3)在肺壁中有表达,到发育的中后期,TGF-β(1β、β2、β3)主要表达于肺间质细胞处。结论TGF-β(1β、2β、3β)在蝌蚪肺发育过程中的阳性反应呈间歇性变化,表明TGF-β(β1、β2、β3)在花背蟾蜍肺发育的不同阶段具有不同的表达特征,可能发挥着不同的功能,它们对花背蟾蜍肺内网状隔膜的形成,肺泡上皮细胞的成熟分化都有重要的作用。     

Amphiregulin-dependent mucous cell metaplasia in a model of nonallergic lung injury

Proliferation and differentiation of the pulmonary epithelium after injury is a critical process in the defense against the external environment. Defects in this response can result in airway remodeling, such as mucus cell metaplasia (MCM), commonly seen in patients with chronic lung disease. We have previously shown that amphiregulin (AREG), a ligand to the epidermal growth factor receptor (EGFR), is induced during the repair/differentiation process elicited by naphthalene-induced lung injury. Thus, we hypothesized that AREG signaling plays an important role in epithelial proliferation and differentiation of the repairing airway. Mice deficient in AREG and lung epithelial EGFR were used to define roles for AREG-dependent EGFR signaling in airway repair and remodeling. We show that AREG and epithelial EGFR expression is dispensable to pulmonary epithelial repair after naphthalene-induced lung injury, but regulates secretory cell differentiation to a mucus-producing phenotype. We show that the pulmonary epithelium is the source of AREG, suggesting that naphthalene-induced MCM is mediated through an autocrine signaling mechanism. However, induction of MCM resulting from allergen exposure was independent of AREG. Our data demonstrate that AREG-dependent EGFR signaling in airway epithelial cells contributes to MCM in naphthalene-induced lung injury. We conclude that AREG may represent a determinant of nonallergic chronic lung diseases complicated by MCM.     

Spatiotemporal expression of flk-1 in pulmonary epithelial cells during lung development

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.     

Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs.

Histologic preparations of lungs form 1-, 5-, 10-, 18-, and 25-day-old rats and adult rats were probed immunohistochemically with specific antibodies for the distribution of epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF). Immunoperoxidase staining of sections of adult rat lungs with a rabbit polyclonal antibody to bovine EGF was strong in ciliated cells of airways and mast cells, nonciliated cells of bronchioles, smooth muscle, type II cells of alveoli, and interstitial and epithelial cells of alveolar septal regions. Developing postnatal lungs had moderate and somewhat diffuse immunoreactivity in all epithelial cells, and more intense staining in vascular smooth muscle. Immunoperoxidase staining of adult rat lungs with a rabbit polyclonal antibody against bovine aFGF was distributed in identical fashion to EGF, with the exception of mast cells which were not reactive. These same sights were localized using an immunoperoxidase sequence with a rabbit polyclonal antibody to the leu 60-leu 98 fragment of aFGF (aFGFfr) with less background. In postnatal developing lungs, immunoreactivity with aFGF and aFGFfr preparations was diffuse and moderately intense in epithelial cells and vascular smooth muscle. Immunoperoxidase staining of adult rat lung sections with a monoclonal antibody to bovine bFGF was strong in hyaluronidase-digested preparations. Reactivity was principally confined to alveolar and vascular basement membrane regions and external laminae of smooth muscle. In early postnatal development, immunoreactivity for bFGF was found in basement membranes beneath developing epithelium and the endothelium of vessel wall intima and in the adventitia, with reactivity increasing with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometry and allometry of the postnatal lung development in the quokka wallaby (Setonix brachyurus): a light microscopic study

The postnatally developing lungs of the quokka wallaby, Setonix brachyurus, were investigated macroscopically and by light microscopic morphometry. Lung, parenchymal and non-parenchymal volumes as well as the components of the latter two were analysed by regression analysis. The lungs comprised a single undivided left lung and a right lung with an adherent accessory lobe. Septal tissue growth was most remarkable in the canalicular and saccular stages. Between mid-canalicular stage and the saccular stage, the lung volume increased 2-fold, mainly due to airspace expansion, coupled with septal tissue thinning. The non-parenchymal vascular volume increase accelerated in the successive developmental stages while the airway and connective tissue volumes progressed in a decreasing order, being highest in the canalicular and saccular stages and lowest in the alveolar stage. Growth and remodelling of the alveolar septa occurred simultaneously with airspace subdivision. Airspace expansion accelerated during the stage of microvascular maturation, when most other parameters showed the least rate of increase.     

Analysis of intrapulmonary vessels and epithelial-endothelial interactions in the human developing lung

The establishment of a sufficiently wide and functional blood-gas interface is of critical importance in lung development, but development of the intrapulmonary vascular system including alveolar capillary vessels still remains unclear. In this study, we first characterized the structural development of the vascular system in accordance with that of airways in human fetal lungs at the pseudoglandular phase (8, 13, and 16 weeks gestation) by examining the immunohistochemical distribution of CD34 and alpha-smooth muscle actin (SMA). Using double immunohistochemistry and 3-dimensional reconstruction techniques, endothelial cells in the developing lung could be classified into two different types according to the characteristics of their adjacent cells (presence or absence of SMA-positive cells) and their distribution (proximal or distal lung parenchyme). Endothelial cells without SMA-positive cells developed into a capillary network surrounding the budding components of distal airways during the mid-pseudoglandular phase before communicating with proximal vessels. We then examined the immunoreactivity of thrombomodulin and von Willebrand factor (vWF) in endothelial cells. Endothelial cells of the capillary network were mainly positive for vWF during the early gestational stages, but altered their phenotypes to those of mature lungs (vWF negative and thrombomodulin positive) during the terminal sac phase. We subsequently determined the immunohistochemical distribution of vascular endothelial growth factor (VEGF). Epithelial cells of the most distal airways were intensely positive for VEGF. These results suggest that VEGF present in airway epithelial cells is involved in the maturation as well as proliferation of capillary endothelial cells. Epithelial-endothelial interactions during lung development are considered very important in the establishment of the functional blood-gas interface.     

IL-1beta disrupts postnatal lung morphogenesis in the mouse

Pulmonary inflammation and increased production of the inflammatory cytokine IL-1beta are associated with the development of bronchopulmonary dysplasia (BPD) in premature infants. To study the actions of IL-1beta in the fetal and newborn lung in vivo, we developed a bitransgenic mouse in which IL-1beta is expressed under conditional control in airway epithelial cells. Perinatal pulmonary expression of IL-1beta caused respiratory insufficiency that was associated with increased postnatal mortality. While intrauterine growth of IL-1beta-expressing mice was normal, their postnatal growth was impaired. IL-1beta disrupted alveolar septation and caused abnormalities in alpha-smooth muscle actin and elastin deposition in the septa of distal airspaces. IL-1beta disturbed capillary development and inhibited the production of vascular endothelial growth factor in the lungs of infant mice. IL-1beta induced the expression of CXC chemokines KC (CXCL1) and macrophage inflammatory protein-2 (CXCL2) and of CC chemokines monocyte chemotactic protein (MCP)-1 (CCL2) and MCP-3 (CCL7), consistent with neutrophilic and monocytic infiltration of the lungs. IL-1beta caused goblet cell metaplasia and bronchial smooth muscle hyperplasia. Perinatal expression of IL-1beta in epithelial cells of the lung caused a lung disease that was clinically and histologically similar to BPD.     

Functional overexpression of wild-type p53 correlates with alveolar cell differentiation in the developing human lung

At 15 weeks after conception (a.c.), the human pulmonary acinus is lined by distal low-columnar and more proximal cuboidal cells that are successive stages in alveolar type II cell differentiation (pseudoglandular period of lung development). From 16 weeks a.c. onward, there are also 'flatter' cells that are intermediate stages in the differentiation of cuboidal type II cells into squamous type I cells (canalicular period). We investigated the role of wild-type p53 protein and the proliferation marker Ki-67 in the differentiation of type II and type I cells in these two periods. Serial sections from fetal lungs (n = 30) were immunoincubated with antibodies against p53 and Ki-67. The presence of prospective type II and type I cells was confirmed using immunohistochemistry for surfactant protein SP-A as a differentiation marker and light and electron microscopy. The p53 and Ki-67 positive nuclei were quantified per alveolar cell phenotype (i.e., low-columnar; cuboidal; flatter). The occurrence of cell apoptosis was studied using propidium iodide (PI) and 4',6'-diamino-2-phenylindol dihydrochloride (DAPI) staining. The combined increase in p53 expression and decrease in Ki-67 expression during alveolar epithelial cell differentiation suggests that wild-type p53 protein plays a role in the differentiation of alveolar type II and type I cells in the human lung, and that this function is mediated through cell cycle arrest. The rare incidence of apoptotic nuclei in alveolar type II cells, together with their absence in alveolar type I cells, supports the view that p53 is involved in the differentiation, rather than the death, of alveolar epithelial cells.