肺泡Ⅱ型上皮细胞转分化

哺乳动物肺泡上皮细胞主要由肺泡Ⅱ型上皮细胞（AECⅡ）和肺泡Ⅰ型上皮细胞（AECⅠ）组成。在肺发育和肺损伤修复过程中,AECⅡ可转分化为AECⅠ,体外原代培养的AECⅡ有这种转分化的特性。现对AECⅡ转分化的标志、影响及调控因素及其在肺损伤中的作用进行综述。     

与成纤维细胞共培养的肺泡II型上皮细胞的生物学特性

建立胎鼠肺泡II型上皮细胞(AECII)与肺成纤维细胞(LF)共培养模型,观察与LF共培养下AECII的生物学特性。倒置相差显微镜观察AECII形态和基本生长情况;RT-PCR和流式细胞术分别检测肺泡表面活性蛋白-C(SP-C)、水通道蛋白5(AQP5)mRNA及蛋白质表达;流式细胞术检测细胞周期及Ki67表达。结果显示,与LF共培养时,AECII能较好地保留其细胞形态,SP-CmRNA及其蛋白质表达明显增加,而AQP5mRNA及其蛋白质表达则明显减少;LF促进AECII增殖,使G2/M、S期细胞及表达Ki67 细胞的比率明显增多。结果提示,AECII与LF共培养时,能更好地保留其细胞形态、分化及增殖特性。     

维甲酸对高氧暴露下原代培养的胎鼠肺泡II型上皮细胞和成纤维细胞增殖与凋亡的影响

探讨高氧暴露对原代培养的胎鼠肺泡II型上皮细胞(AECII)、成纤维细胞(LFs)增殖和凋亡的影响以及维甲酸(RA)的保护作用机制。通过建立高氧暴露原代培养的胎鼠AECII和LFs模型,以RA作为干预方式,采用流式细胞术(膜联蛋白V-PI双标记)检测AECII和LFs凋亡,West-ern印迹检测AECII增殖细胞核抗原(PCNA)、p53及caspase-3表达和LFsPCNA表达。结果发现:(1)与空气对照比较,高氧暴露12h,膜联蛋白V( )PI(-)和膜联蛋白V( )PI( )标记AECII数均显著升高(14.41±1.15vs2.80±0.19,P<0.01;61.07±3.06vs1.49±0.11,P<0.01);RA对空气暴露下AECII坏死、凋亡无明显影响,但明显下调高氧暴露下膜联蛋白V( )PI(-)和膜联蛋白V( )PI( )标记AECII数(8.04±0.79vs14.41±1.15,P<0.01;27.57±2.32vs61.07±3.06,P<0.01)。(2)高氧、RA对LFs坏死、凋亡无明显影响。(3)高氧暴露12h,明显降低AECIIPCNA表达(P<0.01),显著提高其p53(P<0.01)和caspase-3活性片段(P<0.01)表达;RA显著上调高氧暴露下AECIIPCNA表达(P<0.01),下调其p53和caspase-3活化片段表达(P<0.01)。(4)高氧、RA对LFsPCNA表达无明显影响。由此提示,高氧暴露,导致AECII大量凋亡、坏死,增殖受到抑制,同时,LFs所受影响较小,两种细胞对高氧暴露的差异性行为可能是导致未成熟肺组织异常重构的重要原因;RA通过降低AECII凋亡、坏死从而对高氧肺损伤具有保护作用。     

不同浓度TGF-β1对肺泡II型细胞系MLE-12表型及功能的影响

探讨不同浓度及不同时间点TGF-β1对肺泡II型上皮细胞（AECII）表型及功能的影响。小鼠肺泡II型细胞系MLE-12,随机分为：对照组（0ng／mL）、低浓度组（0．1ng／mL）、中浓度组（1ng／mL）和高浓度组（10ng／mL）。应用细胞免疫荧光双标法及荧光定量PCR法观察各组12,24,48,72h细胞形态变化、AECII标记（肺表面活性物质蛋白B,SP—B）及成纤维细胞标记（成纤维细胞特异性蛋白1,FSP—1）蛋白及mRNA的表达情况。结果表明,随着TGF—β1干预时间的延长及浓度的升高,AECII逐渐由鹅卵石样变成纺锤体形状,获得成纤维细胞样外观。蛋白水平,AECII标记SP-B表达逐渐减弱,成纤维细胞标记FSP-1表达逐渐增强,48h中浓度组及24h高浓度组两者可见明显的共表达,同时,其SP-BmRNA表达较同时间对照组下调,而FSP1 mRNA表达较同时间对照组上调。低浓度组各时间点上述表现不明显。TGF—β1促使AECII向成纤维细胞转化（EMT）,且具有时间及浓度依赖性。     

肺泡Ⅱ型上皮细胞自噬体与结核分枝杆菌相互作用的研究

目的:检测LC3在肺泡Ⅱ型上皮细胞A549上的表达情况,及结核分枝杆菌刺激后对其表达的影响,探讨自噬在结核分枝杆菌感染上皮细胞中所起的作用。方法:体外培养肺泡Ⅱ型上皮细胞A549,在结核分枝杆菌感染A549细胞0h,24h分别提取RNA,采用RT-PCR的方法检测LC3mRNA的表达情况。采用凋亡坏死染色试剂盒在结核分枝杆菌感染24h后检测对照组,3-MA组,MTB组和3-MA+MTB组的细胞坏死情况。在结核分枝杆菌感染A549细胞4h,8h,16,24h采用Non-Radioactive Cytocity Assay的方法检测对照组,3-MA组,MTB组和3-MA+MTB组上清液LDH的OD值。结果:LC3在肺泡Ⅱ型上皮细胞显著表达,结核分枝杆菌感染后LC3表达降低。细胞凋亡和坏死染色结果显示空白组和3-MA组没有明显差异(P>0.05),MTB组和3-MA+MTB组有明显差异(P<0.05)。LDH检测显示MTB组和3-MA+MTB组上清液LDH的OD值数据两两之间有明显差异(P<0.05)并且有时间依赖性。结论:肺泡II型上皮细胞自噬体在抵抗结核分枝杆菌的感染过程中起一定的作用。     

周期性机械牵张对肺泡Ⅱ型上皮细胞Cyr61表达的影响

目的研究周期性牵张肺泡Ⅱ型上皮细胞株A549细胞对Cyr61表达的影响。方法对肺泡Ⅱ型上皮细胞株A549细胞施加周期性机械牵张应力。加载频率0.5 Hz,加载时间2 h,加载应力分别为5%,15%,30%。加载应力为15%,加载频率0.5 Hz,加载时间分别0,15 min,30 min,60 min,120 min。每个实验均设立空白对照即不给予机械应力。用PCR法测定Cyr61 mRNA的表达,用western法测定Cyr61蛋白含量。结果随着加载应力的增加和加载时间的延长,Cyr61蛋白含量和mRNA表达均增加(P<0.05);施加不同加载幅度后,Cyr61蛋白含量和mRNA表达均增加(P<0.05)。结论肺泡Ⅱ型上皮细胞IL-8的产生和释放与周期性的机械牵张应力呈强度和时间依赖性。     

降钙素基因相关肽通过增加自噬对抗新生大鼠氧化应激性肺损伤

本研究组前期研究显示,降钙素基因相关肽(calcitonin gene-related peptide, CGRP)对高氧诱导的氧化应激性肺损伤具有保护作用。本研究旨在探讨细胞自噬是否参与CGRP对新生大鼠氧化应激性肺损伤的保护作用。新生Sprague-Dawley (SD)大鼠随机分为5组：空气对照组(Control组)、高氧损伤模型组(Model组)、CGRP干预组(Model+CGRP组)、Model+CGRP+自噬激动剂组(Model+CGRP+Rapamycin组)和Model+CGRP+自噬抑制剂组(Model+CGRP+LY294002组)。采用新生SD大鼠持续吸氧(吸入氧浓度百分比FiO2为90%～95%)14天的方法制备氧化应激性肺损伤模型。苏木精-伊红(HE)染色观察肺组织病理变化并测定肺泡平均线性截距(mean linear intercept, MLI)。透射电镜观察肺泡II型上皮细胞(type II alveolar epithelial cell,AECII)内自噬囊泡数量改变。Western blot检测肺组织裂解液中Caspase-3、Bcl-2、mTO...     

经气管灌注血管紧张素Ⅱ导致凋亡性急性肺损伤(英文)

为研究外源性血管紧张素Ⅱ（angiotensinⅡ,ANG）在急性肺损伤和肺泡上皮细胞凋亡中的作用,经气管分别给雄性Wistar大鼠（175-200g）灌注ANG、ANG加caspase抑制剂ZVAD—fmk、ANG加ANG受体1阻断剂losartan和仅灌注磷酸盐缓冲溶液（PBS）。6或20h后在体灌洗动物肺脏,测定灌洗液中血红蛋白（hemoglobin,Hb）和荧光物质（BODIPY）标定的白蛋白含量（在灌洗前15min静脉注入BODIPY-白蛋白）。TUNEL测定显示,灌注ANG6h后,支气管和肺泡上皮细胞内标定的DNA片断显著增2H（P〈0．05）;ANG所致的DNA片断增加可被同时灌注ZVAD—fmk或losartan阻断。灌注ANG后免疫标定caspase3阳性细胞数量显著增多（P〈0．01）,ZVAD—fmk或losartan同样显著减少caspase3阳性细胞的数量。灌注ANG显著增加肺泡灌洗液中荧光标定的白蛋白（P〈0．01）和Hb的含量（P〈0．05）;ZVAD—fmk或losartan亦显著抑制荧光白蛋白和Hb含量的变化。结果表明,肺泡上皮细胞在体暴露于外源性ANG足以引起ANG受体1介导的上皮细胞凋亡和肺泡屏障损伤。     

上皮细胞转分化的信号转导研究进展及其在肾小管的研究现状

上皮细胞在特定生理和病理情况下向间充质细胞转分化的现象是肿瘤及慢性炎症性疾病中的重要细胞生物学现象。近年来研究发现 ,细胞内信号转导途径中的MAPK通路、Rho家族激酶、Src激酶、PI3激酶和Smad通路参与调控上皮细胞转分化过程。肾小管上皮细胞在病理条件下可转分化为肌成纤维细胞 ,其细胞内信号转导机制虽已有初步研究 ,但尚所知甚少。本文将就有关的研究进展及现状进行综述     

放射性肺损伤细胞渗出及SP-A表达的实验研究

目的探讨急性放射性肺损伤不同时间点病理和肺泡灌洗液与肺泡表面活性蛋白A(SP-A)表达的变化。方法健康雄性Wistar大鼠40只随机分为对照组(C组)和照射组(R组)。行6MV-X线全胸野照射,剂量率2Gy/min,单次剂量15Gy,源皮距1m,照射面积4.5cm×4.5cm。于照射后第1,2,4,8周取肺组织作HE、Masson染色,肺泡灌洗液进行细胞计数,蛋白免疫印迹(Westernblot)检测肺组织中SP-A蛋白表达。结果HE和Masson染色提示照射后的第1周始肺泡腔有炎性细胞渗出,继之间质水肿,第4及8周出现肺泡腔变小甚至结构破坏,局部实变,肺间质出现胶原纤维;肺泡灌洗液的细胞总数照射组与对照组相比各时间段都明显增高(P<0.01);照射组各时间点SP-A蛋白表达均明显下降(P<0.001),以第1、8周下降最为明显,第2、4周出现回升。结论SP-A蛋白参与放射性肺损伤的发生、发展过程,为进一步探讨放射性肺损伤的发病机制提供了实验依据。     

Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls (P<0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.     

角质细胞生长因子的研究进展

角质细胞生长因子(KGF)是成纤维细胞生长因子(FGFs)家族的成员,即FGF-7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26—28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。KGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

Glycochenodeoxycholate induces rat alveolar epithelial type II cell death and inhibits surfactant secretion in vitro

Bile acid-induced lung injury has become an important topic for neonatologists after the discovery of a high incidence of infant respiratory distress syndrome complicated from maternal intrahepatic cholestasis. To explore the molecular pathway of bile acid-induced lung injury, we investigated the cytotoxicity of the glycochenodeoxycholate (GCDC) to alveolar epithelial type II cells (AECII), as the main component of bile acid. The results demonstrated that glycochenodeoxycholate induced oxidative stress, mitochondrial damage, and increased caspase activity in the primary cultured AECII. Moreover, ROS scavengers and caspase inhibitors could rescue cell death induced by GCDC in rat AECII. Our results also indicated that GCDC inhibited AECII surfactant secretion. In conclusion, this study suggested that cell death prevention and cell therapy should be considered as therapeutic strategies for infant respiratory distress syndrome complicated from maternal intrahepatic cholestasis.     

Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO(2) and PCO(2). Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O(2)-5% CO(2)) or hypercapnia (21% O(2-) 15% CO(2)) and expression of surfactant protein C was measured and compared to normal conditions (21% O(2)- 5% CO(2)). Cell cycle progression and apoptosis were measured by flow cytometric analysis. RESULTS: A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-alpha partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. CONCLUSIONS: Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.     

IL10 deficiency promotes alveolar enlargement and lymphoid dysmorphogenesis in the aged murine lung

The connection between aging‐related immune dysfunction and the lung manifestations of aging is poorly understood. A detailed characterization of the aging IL10‐deficient murine lung, a model of accelerated aging and frailty, reconciles features of both immunosenescence and lung aging in a coherent model. Airspace enlargement developed in the middle‐aged (12 months old) and aged (20–22 months old) IL10‐deficient lung punctuated by an expansion of macrophages and alveolar cell apoptosis. Compared to wild‐type (WT) controls, the IL10‐deficient lungs from young (4‐month‐old) mice showed increased oxidative stress which was enhanced in both genotypes by aging. Active caspase 3 staining was increased in the alveolar epithelial cells of aged WT and mutant lungs but was greater in the IL10‐deficient milieu. Lung macrophages were increased in the aged IL10‐deficient lungs with exuberant expression of MMP12. IL10 treatment of naïve and M2‐polarized bone marrow‐derived WT macrophages reduced MMP12 expression. Conditioned media studies demonstrated the secretome of aged mutant macrophages harbors reduced AECII prosurvival factors, specifically keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), promotes cell death, and reduces survival of primary alveolar epithelial cells. Compared to WT controls, aged IL10‐deficient mice have increased parenchymal lymphoid collections comprised of a reduced number of apoptotic cells and B cells. We establish that IL10 is a key modulator of airspace homeostasis and lymphoid morphogenesis in the aging lung enabling macrophage‐mediated alveolar epithelial cell survival and B‐cell survival within tertiary lymphoid structures.     

IL-33-ST2 pathway regulates AECII transdifferentiation by targeting alveolar macrophage in a bronchopulmonary dysplasia mouse model

Evidence points to the indispensable function of alveolar macrophages (AMs) in normal lung development and tissue homeostasis. However, the importance of AMs in bronchopulmonary dysplasia (BPD) has not been elucidated. Here, we identified a significant role of abnormal AM proliferation and polarization in alveolar dysplasia during BPD, which is closely related to the activation of the IL-33-ST2 pathway. Compared with the control BPD group, AMs depletion partially abolished the epithelialmesenchymal transition process of AECII and alleviated pulmonary differentiation arrest. In addition, IL-33 or ST2 knockdown has protective effects against lung injury after hyperoxia, which is associated with reduced AM polarization and proliferation. The protective effect disappeared following reconstitution of AMs in injured IL-33 knockdown mice, and the differentiation of lung epithelium was blocked again. In conclusion, the IL-33-ST2 pathway regulates AECII transdifferentiation by targeting AMs proliferation and polarization in BPD, which shows a novel strategy for manipulating the IL-33–ST2-AMs axis for the diagnosis and intervention of BPD.     

The Hippo–YAP pathway regulates the proliferation of alveolar epithelial progenitors after acute lung injury

Regeneration of pulmonary epithelial cells plays an important role in the recovery of acute lung injury (ALI), which is defined by pulmonary epithelial cell death. However, the mechanism of the regenerative capacity of alveolar epithelial cells is unknown. Using a lung injury mouse model induced by hemorrhagic shock and lipopolysaccharide, a protein mass spectrometry‐based high‐throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells (AEC2s), we analyzed the mechanism of alveolar epithelial cells proliferation. We demonstrated that the expression of Hippo‐yes‐associated protein 1 (YAP1) key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI. Furthermore, the results showed that YAP1+ cells in lung tissue after ALI were mainly Sftpc lineage‐labeled AEC2s. An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 small interfering RNA and Hippo inhibitor. These findings revealed that YAP functioned as a key regulator to promote AEC2s proliferation, with the Hippo signaling pathway playing a pivotal role in this process.