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     

人胚胎肺的组织发生

本文将38例胎儿肺组织,用Romeis法显示弹性纤维,PAS法显示糖原,Unna法显示RNA及Gomori钙-钴法显示ALP。胚胎期肺的发育可分为3期:1.腺状期(7～16周)以支气管树发育为主,上皮为假复层柱状,含有丰富的糖原,上皮游离缘有明显的嗜哌喏宁及ALP阳性闭锁堤;2.小管期(17～24周)以呼吸部发育为主,衬以未分化的立方上皮,上皮内糖原逐渐减少;3.原始肺泡期(25周至出生)以肺泡的形成和分化为主,肺泡上皮分化为扁平的Ⅰ型细胞及立方形Ⅱ型细胞。Ⅱ型细胞含有少量糖原颗粒及RNA颗粒,ALP弱阳性。此期有大量毛细血管嵌入上皮间,形成龛细胞及气血屏障。     

胎肺发育分化中表皮生长因子受体的表达和作用

目的　观察表皮生长因子受体 (EGFR)在小鼠胎肺发育过程的表达特征 ,研究表皮生长因子 (EGF)和转化生长因子 α(TGF α)通过与EGFR的作用 ,对胎肺形态发生和肺泡上皮成熟分化的作用。方法　昆明小鼠 ,13- 19天胎肺组织、生后1、7、14、30天及成鼠肺组织共 12组 ,常规石蜡切片 ,采用免疫组化SP两步法检测EGFR的表达。结果　胎肺假腺期 ,EGFR的表达主要定位于支气管呼吸道上皮 ,小管期EGFR阳性反应达到最高峰。在原始肺泡期 ,阳性反应主要在肺泡上皮细胞。出生后 ,支气管上皮细胞EGFR表达重新呈阳性表达。结论　在胎肺发育的不同时期 ,EGFR在上皮细胞的定位有迁移 ,免疫组化反应强弱也有差异 ,说明EGFR在胎肺不同发育阶段发挥不同的功能 ,它不仅参与支气管树的形成 ,还对呼吸道上皮细胞和肺泡上皮细胞的成熟分化有重要调节作用。     

生长因子对Ⅱ型肺泡上皮细胞增生的调控

Ⅱ型肺泡上皮细胞（简称Ⅱ型细胞）在肺损伤后肺泡上皮的修复与更新以及肺发育过程中发挥重要作用，生长因子是Ⅱ型细胞增殖、分化的重要调节因子，本文就有关对Ⅱ型细胞起调控作用的主要生长因子研究进展作一概述。     

小鼠胎肺发育中肺泡上皮细胞的电镜观察

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 ,细胞之间有连接复合体。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 型肺泡细胞 ,其特征为表面有微绒毛 …     

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。     

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。     

胎儿肺泡上皮和气血屏障的电镜观察

为阐明不同胎龄肺泡表面上皮和气血屏障的发育状况,对24例受精龄为14～40周的人胎肺脏,进行透射电镜观察。胎龄19周时,即可辨认肺泡Ⅰ型上皮细胞和气血屏障。图像分析表明,气血屏障的厚度,随胎龄的增长而变薄。Ⅱ型肺泡上皮细胞,在胎龄20周后逐渐分化,22周时胞质内出现板层小体,小体的截面积随胎龄而增大。本文着重对气血屏障及Ⅱ型上皮细胞的形态变化进行了讨论。     

胎儿肺泡Ⅱ型细胞超微结构特点

目的探讨肺泡Ⅱ型上皮细胞发生过程中超微结构特点。方法利用电子显微镜技术观察20例不同胎龄肺泡Ⅱ型细胞板层小体及细胞器的变化。结果胎龄14～18w肺泡上皮呈单层柱状，不能分辨Ⅰ、Ⅱ型细胞，自25～40wⅡ型细胞板层小体逐渐增多。平均截面积由4．15＋０．84增至18．23＋1．12（μm^2）；它们与胞质比值相对增加。结论胎龄19w以后Ⅱ型细胞板层小体逐渐形成和增多，其截面积与胞质比例随胎龄增多。     

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。     

An ultrastructural, morphometric analysis of rabbit fetal lung type II cell differentiation in vivo

Rabbit lung type II cell differentiation was evaluated by use of ultrastructural, morphometric techniques. Fetal lung epithelial cells decreased in size dramatically from day 19 to day 21 of gestation. Thereafter, the cell and cytoplasmic cross-sectional area declined gradually until the neonatal time point. The tall columnar cell shape characteristic of fetal lung epithelial cells at early stages of development became cuboidal by day 24 of gestation. The number of mitochondria per micron2 cytoplasmic area in presumptive alveolar epithelial cells and the mitochondrial volume density increased toward the end of gestation. The volume density of glycogen pools within fetal lung epithelial cells reached a plateau on day 21 of gestation and then declined sharply on day 26 of gestation in lamellar body-containing, type II epithelial cells. Lamellar bodies increased in number and volume density in epithelial cells starting on day 26 of gestation and peaked with respect to these parameters in the neonatal lung tissue. Multivesicular bodies, which are thought to be a precursor to the lamellar body, became more prominent in differentiated type II cells on day 26 of gestation and increased in volume density from day 28 of gestation to the adult time point. The distance between mesenchymal and epithelial cells in fetal lung tissue declined sharply between days 24 and 26 of gestation but remained relatively constant thereafter. Foot processes extending from connective tissue cells contiguous to the epithelium were generally more numerous than those extending from the basal plasma membrane of epithelial cells at every stage of development examined. These data quantitate for the first time key ultrastructural events that occur during the differentiation of fetal lung epithelial cells in vivo.     

The maturation of the rabbit fetal lung following maternal administration of pilocarpine

Pilocarpine HCl, a parasympathomimetic drug, was administered to pregnant white rabbits in a daily subcutaneous injection of 5 mg/kg on days 24 through 27 of gestation. Fetuses from these animals and from salineinjected controls were obtained by caesarean section at day 28 of gestation. Light microscopic examination revealed thinner alveolar septa in the lungs of pilocarpine-treated fetuses and, morphometrically, a significant increase in the number of mature type II cells, both per unit area and per 1,000 lung cells of any kind. Examination by electron microscopy revealed that the alveolar epithelium of pilocarpine-treated fetuses demonstrated morphologic correlates of increased maturation. These included thinning of type I cells to form blood-air barriers and substantial reductions in the glycogen content of both epithelial cell types. Type II cells of pilocarpine-treated fetuses contained (as indicated by morphometric analysis) more and larger lamellar inclusion bodies, as well as more multivesicular bodies than those of controls. Biochemical determination indicated that the glycogen content of fetal lung, but not liver, was reduced significantly in the pilocarpine-treated group. The findings of this study indicate that maternal administration of pilocarpine results in increased maturation of the fetal alveolar epithelium, thus providing a basis for the autonomic manipulation of fetal lung maturation.     

肺泡上皮细胞在肺部免疫中的作用

肺泡是肺脏进行气体交换的基本单位,其内表面覆盖着Ⅰ型和Ⅱ型肺泡上皮细胞。肺泡上皮细胞在面积上的绝对优势使其相较于肺泡巨噬细胞,更有机会在第一时间和病原体直接接触。肺泡上皮除了形成致密的屏障以隔绝外源性致病原外,也通过其表面受体和分泌产物与免疫细胞相互作用,来维持肺部的稳态和相对无菌性。肺上皮异常损伤和过度修复与多种肺部疾病如慢性阻塞性肺病、肺纤维化直接相关。本文综述了关于肺泡上皮细胞在屏障作用、病原防御、调节免疫功能以及和肺部疾病关系的研究进展。     

Hypophysectomy and porcine fetal lung development

The effect of hypophysectomy on the development of the lung parenchyma and maturation of pulmonary alveolar type II cells was examined in the fetal pig. In fetuses from four different gilts, hypophysectomy was performed on gestational day 69 or 70. Littermates from the same gilts served as controls. Fetuses were delivered by caesarean section at term (113 +/- 1 days of gestation), and the lungs were fixed by intratracheal instillation. Plasma cortisol and thyroxine concentrations in the umbilical artery were markedly lower for hypophysectomized fetuses compared with control fetuses. Body weight was similar for both groups of fetuses. Total lung volume was 53% smaller in hypophysectomized fetuses compared with control fetuses. However, alveolar septal tissue and capillary luminal volumes were similar in both groups. Total lung alveolar surface area was twice as great in control animals compared with hypophysectomized animals. The volumes of epithelium, interstitium, and endothelium of centriacinar alveolar septa per unit surface area of epithelial basal lamina were 3.7, 4.8, and 2.4 times greater in hypophysectomized fetuses compared with control fetuses. Alveolar type II cell composition also differed significantly between groups. The volume fraction of glycogen in type II cells was 51% for hypophysectomized fetuses and 12% for control fetuses, while lamellar body volume fraction was 8% in hypophysectomized fetuses and 23% in control fetuses. The frequency of alveolar type II cell contact with mesenchymal interstitial cells via foot processes was 5 times greater in the lungs of control animals compared with hypophysectomized animals. These findings demonstrate significant effects of hypophysectomy on the morphogenetic and cytodifferentiation activities of all major tissue compartments of the pulmonary gas exchange area during the final trimester of fetal development.     

L-azetidine-2-carboxylic acid retards lung growth and surfactant synthesis in fetal rats

Maturation of the pulmonary epithelium during late fetal development is controlled at least in part by the underlying fibroblasts. To further investigate this cellular interdependence and the role of collagen in type 2 cell differentiation, we studied the effects of inhibiting fibroblast function in vivo by injecting the proline analog L-azetidine-2-carboxylic acid (LACA) to timed pregnant rats, and examining changes in cell proliferation and surfactant synthesis in fetal lungs. LACA (200 mg/kg) was injected twice daily for 2 days and rats were killed 2 days later at days 19, 20, 21, and 22 of gestation. Fetal lung weight and DNA content were about 50% of controls, hydroxyproline per dry weight was reduced and by electron microscopy, there appeared to be less fibrillar collagen in the lung. Autoradiography after [3H]thymidine pulse-labeling showed reduced cell proliferation on days 19 and 20 mainly due to lower fibroblast growth with a smaller reduction in epithelial labeling. Lung development in LACA-treated rats was retarded; air sacs were slow to open, epithelial cells retained glycogen longer and fewer cells developed lamellar bodies compared with age-matched controls. There was a reduction in the incidence of epithelial-interstitial cell contacts at day 20 only. Measurements of disaturated phosphatidylcholine showed a 50% reduction per dry weight and a lower disaturated phosphatidylcholine/lipid ratio after LACA. The results indicate that LACA administration in vivo slows fibroblast growth and greatly reduces fibrillar collagen deposition with an accompanying reduction in pulmonary surfactant. This suggests that secreted matrix influences growth and differentiation of the alveolar epithelium.     

Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.

Pulmonary surfactant is a lipoprotein substance, comprised of approximately 80% phospholipid and approximately 10% protein, that lowers surface tension at the air-alveolar aqueous interface. Surfactant is synthesized and secreted by alveolar type II epithelial cells where it is stored intracellularly in lamellar bodies. In the present study, we used the technique of in situ hybridization to localize the mRNA for two surfactant-associated proteins, SP-A and SP-B, in developing rabbit fetal lung tissue. We found that SP-A mRNA was first localized in rabbit fetal lung alveolar type II cells on day 26 of gestation, the time at which lamellar bodies are first observed within fetal lung type II cells. On day 28 of gestation, a very small amount of SP-A mRNA was also detectable in the epithelial cells of some bronchioles. In neonatal and adult rabbit lung tissue, SP-A mRNA was primarily restricted to alveolar type II cells; however, the epithelial cells of some bronchioles contained small amounts of SP-A mRNA. SP-B mRNA was first detected in cuboidal epithelial cells in the prealveolar region of the rabbit fetal lung tissue on day 24 of gestation, i.e., at least 2 days before the appearance of SP-A mRNA and lamellar bodies within differentiated alveolar type II cells. SP-B mRNA was detected in most bronchiolar epithelial cells of the rabbit fetal lung tissue at day 28 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

小鼠肺发育中细胞增殖与凋亡及相关调控基因的表达

目的 探讨小鼠肺发育过程细胞增殖与凋亡的变化规律及凋亡相关基因ｐ５３、ｂｃｌ－２ｄ在肺发育中的表达意义。方法 不同发育天数的小鼠肺组织,采用免疫组织化学及ＴＵＮＥＬ法,观察细胞增殖与凋亡及相关基因ｐ５３、ｂｃｌ－２的表达。结果 １．胎１４和１６ｄ肺ＰＣＮＡ阳性细胞数达到高峰;之后ＰＣＮＡ阳性率逐渐下降;生后４到１４ｄ,又恢复低水平的细胞增殖,２．小鼠肺发育过程中出现两个细胞凋亡高峰：即胎１４至１６     

Development of alveolar septa and cellular maturation within the perinatal lung

To quantitate fetal lung cellularity and regional variation in alveolar maturation, guinea pig lungs were studied at 55, 60, and 65 d of gestation or within 2 h of birth (term = 68 d), and the data were analyzed for intralobar, interlobar, and age-group differences. Nine blocks from each left cranial and caudal lobe were all measured for volume, numerical, and surface densities in tissue (Vv(i,t), Nv(i,t), and Sv(i,t], and total volumes, numbers, and surface areas per lung (V, N, and S) of type I and type II epithelia, presumptive progenitor epithelium (cuboidal cells lacking lamellar bodies [LB]), interstitium, and endothelium. Total fixed lung volume, VL, increased 3-fold from day 55 through birth. At each age, there were no consistent intralobar or interlobar differences in Vv(i,t), Nv(i,t), or Sv(i,t) for any cell type. Within a septal tissue volume of 580 to 670 microliters at all ages, the N and V of type I cells did not vary with age, although their S increased from 1,240 cm2 at day 55 to 3,967 cm2 at birth. The N of morphologic type II cells per lung increased 7-fold from day 55 to day 60 and was constant thereafter, while the N of cuboidal cells decreased proportionally; type II cells contained only 4.8% (vol/vol) of LB at 55 d compared to 18.0% at birth. The N and V of interstitium did not vary by age. While endothelial V was constant over these ages, endothelial S increased from 897 cm2 to 3,398 cm2, and the V of capillary blood and the V of alveolar airspace each increased 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fas-ligand-induced apoptosis of respiratory epithelial cells causes disruption of postcanalicular alveolar development

Premature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia- associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)(7)-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA(+)/(TetOp)(7)-FasL(+) mice compared with single-transgenic CCSP-rtTA(+) littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. These data demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis.     

Localization of surfactant-associated protein C (SP-C) mRNA in fetal rabbit lung tissue by in situ hybridization.

Surfactant is a lipoprotein substance that is synthesized and secreted by alveolar type II epithelial cells and acts to reduce surface tension at the air-alveolar interface. SP-C is a 5,000-D molecular weight, hydrophobic, surfactant-associated protein. In the present study, we used a ribonuclease protection assay to show that SP-C mRNA is induced in rabbit fetal lung tissue early in development, increases in relative concentration as development proceeds, and is present in maximal concentration at term (31 days of gestation). We also used the technique of in situ hybridization to localize SP-C mRNA in fetal, neonatal, and adult rabbit lung tissue. SP-C mRNA was present in all of the epithelial cells of the prealveolar region of day 19 gestational age rabbit fetal lung tissue, i.e., about 7 days before the appearance of differentiated alveolar type II cells in the fetal lung tissue. By day 27 of gestation, SP-C mRNA was restricted to epithelial cells with the morphologic characteristics of alveolar type II cells. SP-C mRNA was not detected in bronchiolar epithelium at any stage of lung development. The intensity of SP-C mRNA hybridization in the prealveolar and alveolar type II epithelial cells increased as a function of gestational age and was maximal at term. The pattern of SP-C mRNA localization in neonatal and adult rabbit lung tissue was consistent with the restriction of SP-C gene expression to differentiated alveolar type II cells. Our data are suggestive that SP-C may serve some as yet unknown function early in lung development because it is present in fetal lung prealveolar epithelial cells much earlier in gestation than are differentiated, surfactant-producing alveolar type II cells.