Pulmonary Surfactant-associated Protein Research Articles

GW29-e0814 Genetic polymorphisms of pulmonary surfactant-associated protein A gene may be potential risk factors of severe obstructive sleep apnea

Decreased circulating surfactant protein A (SP-A) was observed in some individuals with higher hypopnea index (HI) in obstructive sleep apnea (OSA) in previous study, the present study aimed to further explore the role of pulmonary surfactant-associated protein A (SFTPA) gene in OSA. In this case-

Effects of aerosol inhalation of recombinant human keratinocyte growth factor 2 on the lung tissue of rabbits with severe smoke inhalation injury

Objective: To investigate the effect of recombinant human keratinocyte growth factor 2 (rhKGF-2) on lung tissue of rabbits with severe smoke inhalation injury. Methods: A total of 120 New Zealand rabbits were divided into 5 groups by random number table after being inflicted with severe smoke inhalation injury, with 24 rats in each group. Rabbits in the simple injury group inhaled air, while rabbits in the injury+phosphate buffer solution (PBS) group inhaled 5 mL PBS once daily for 7 d. Rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group received aerosol inhalation of 1 mg/kg, 2 mg/kg, and 5 mg/kg rhKGF-2 (all dissolved in 5 mL PBS) once daily for 7 d, respectively. On treatment day 1, 3, 5, and 7, blood samples were taken from the ear central artery of 6 rabbits in each group. After the blood was taken, the rabbits were sacrificed, and the tracheal carina tissue and lung were collected. Blood pH value, arterial oxygen partial pressure (PaO(2)), arterial blood carbon dioxide pressure (PaCO(2)), and bicarbonate ion were detected by handheld blood analyzer. The expressions of pulmonary surfactant-associated protein A (SP-A) and vascular endothelial growth factor (VEGF) in lung tissue were detected by Western blotting. Pathomorphology of lung tissue and trachea was observed by hematoxylin-eosin staining. Data were processed with analysis of variance of two-way factorial design and Tukey test. Results: (1) Compared with those in simple injury group, the blood pH values of rabbits in the latter groups on treatment day 1-7 had no obvious change (P>0.05). The PaO(2) of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were (75.0±2.4) and (71.0±4.5) mmHg (1 mmHg=0.133 kPa), respectively, which were significantly higher than (62.0±6.8) and (63.0±3.0) mmHg in simple injury group (q=4.265, 8.202, P 0.05). The expressions of SP-A in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group on treatment day 3 were 0.091±0.007 and 0.101±0.009, respectively, significantly higher than 0.069±0.009 in simple injury group (q=10.800, 13.580, P 0.05). Compared with those in simple injury group, the expressions of VEGF in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 and 7 were significantly increased (q=4.243, 8.000, P<0.05 or P<0.01), and the expression of VEGF in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=20.720, P<0.01). (4) On treatment day 1, the injury of rabbits in each group was similar, with a large number of neutrophils infiltrated and abscess formed in the alveolar and interstitial tissue, thickened alveolar septum, some collapsed alveolar and atelectasis; large area of tracheal mucosa was degenerated and necrotic, with a large amount of inflammatory exudates blocking in the cavity. On treatment day 3, the inflammation of lung tissue and trachea in each group were improved, but the inflammation in simple injury group and injury+PBS group was also serious. On treatment day 5, the inflammation in lung tissue and trachea of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group were improved much obviously than those in the other groups. On treatment day 7, the inflammation in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group alleviated obviously than those in the other groups, most alveoli had no obvious exudative fluid, the alveolar cavity was intact and clear, the local alveolar dilated like a cyst, and the alveolar septum thinning; the improvement of inflammation of trachea was more obvious than the other groups, the tracheal mucosa tended to be more complete, and few neutrophils were infiltrated in the endotracheal cavity. Conclusions: Atomization inhalation of rhKGF-2 can improve the PaO(2) level of rabbits with severe smoke inhalation injury, reduce airway inflammation, increase the expression of SP-A and VEGF in lung tissue, thus promoting the repair of lung tissue.

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes

Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.

Evaluation of pulmonary reexpansion in a porcine model of bronchial foreign body.

This study was designed to establish a porcine model of reversible obstruction of endobronchial foreign body (FB) and to assess the natural pulmonary reexpansion process using radiology and histopathology after removal of the bronchial FB. A metal stent was placed in the right lower lobe bronchial lumen of 15 pigs by bronchoscopy and removed after 2 weeks of stent retention. Animals were divided into two groups (group I and II), based on when they were sacrificed (week 2 or week 4) after stent removal. Pigs underwent computerized tomography scan to document pulmonary radiological changes. The lungs were harvested for electron microscopy examination, and the expression of pulmonary surfactant-associated protein A (SPA) was determined by immunohistochemistry. Thirteen (86.7%) animals successfully responded to the intervention. Atelectasis was formed by stent implantation within 2 weeks. The ratio of the residual atelectasis area was smaller in group II compared to that in group I (P < 0.01). The histological manifestations of experimental lungs were significantly improved in group II. Moreover, the expression of SPA in group II was higher compared to that in group I (P < 0.01). We have established a model of reversible bronchial FB obstruction in pigs that is both feasible and appropriate for evaluating the long-term process of pulmonary reexpansion after removal of FB. Bronchial metal FB retention for a period of 2 weeks could form atelectasis, and a natural pulmonary recovery process related to atelectasis takes approximately 4 weeks after removal of metal FB.

Proteomic diversity of high‐density lipoprotein explains its association with clinical outcome in patients with heart failure

Previously, low high-density lipoprotein (HDL) cholesterol was found to be one of the strongest predictors of mortality and/or heart failure (HF) hospitalisation in patients with HF. We therefore performed in-depth investigation of the multifunctional HDL proteome to reveal underlying pathophysiological mechanisms explaining the association between HDL and clinical outcome. We selected a cohort of 90 HF patients with 1:1 cardiovascular death/survivor ratio from BIOSTAT-CHF. A novel optimised protocol for selective enrichment of lipoproteins was used to prepare plasma. Enriched lipoprotein content of samples was analysed using high resolution nanoscale liquid chromatography-mass spectrometry-based proteomics, utilising a label free approach. Within the HDL proteome, 49 proteins significantly differed between deaths and survivors. An optimised model of 12 proteins predicted death with 76% accuracy (Nagelkerke R2 =0.37, P< 0.001). The strongest contributors to this model were filamin-A (related to crosslinking of actin filaments) [odds ratio (OR) 0.31, 95% confidence interval (CI) 0.15-0.61, P= 0.001] and pulmonary surfactant-associated protein B (related to alveolar capillary membrane function) (OR 2.50, 95% CI 1.57-3.98, P< 0.001). The model predicted mortality with an area under the curve of 0.82 (95% CI 0.77-0.87, P< 0.001). Internal cross validation resulted in 73.3 ± 7.2% accuracy. This study shows marked differences in composition of the HDL proteome between HF survivors and deaths. The strongest differences were seen in proteins reflecting crosslinking of actin filaments and alveolar capillary membrane function, posing potential pathophysiological mechanisms underlying the association between HDL and clinical outcome in HF.

Pulmonary surfactant-associated proteins and inflammatory factors in obstructive sleep apnea.

Pulmonary surfactant (PS) plays roles in promoting the removal of the liquid, host defense, and immune regulation in the tracheal, bronchial, and alveoli epithelium. PS protein expression level can be regulated by oxygen levels and related free radicals. Obstructive sleep apnea (OSA) is featured with oxygen free radical production for damaging epithelial tissues and thus may affect PS production. The study was to explore the relationship between PS protein and OSA severity. We collected serum and bronchoalveolar lavage fluid (BALF) samples from 35 OSA patients and 22 healthy subjects. PS-associated proteins and inflammatory factors, including surfactant proteins, HIF-1α, NF-κB, and IL-6, were analyzed. Regression analysis was performed to reveal the relationship between biochemical factors and clinical indexes recorded during PSG monitor. Lower BALF and surfactant protein (except surfactant protein C or SPC) levels occurred in OSA patients (all p<0.05 compared to control group). A strongly negative correlation was found between surfactant protein with apnea-hypopnea index (AHI) and other sleeping indexes including ODI3 and ODI4. Similar patterns were found in serum samples, which were strongly correlated with BALF counterparts. Surfactant proteins were further found to have negative regression with inflammatory factors such as HIF-1α, NF-κB, and IL-6. This study established the relationship between PS-related protein with severity of OSA, plus their relationship with inflammatory factors. Our results provided possibly novel markers in general circulation for disease evaluation of OSA.

Comparative Proteomic Analysis of Rat Bronchoalveolar Lavage Fluid after Exposure to Zinc Oxide Nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanomaterials in consumer products and industrial applications. As a result of all these uses, this has raised concerns regarding their potential toxicity. We previously found that candidate markers of idiopathic pulmonary fibrosis and lung cancer were significantly up-regulated in rat bronchoalveolar lavage fluid (BALF) following exposure to ZnO NPs by using a liquid chromatography (LC)-based proteomic approach. To achieve comprehensive protein identification analysis, we conducted the two-dimensional gel electrophosis (2-DE)-based proteomic workflow to analyze the differences in BALF proteins from rats that had been exposed to a high dose of 35 nm ZnO NPs. A total of 31 differentially expressed protein spots were excised from the gels and analyzed by nanoLC-tandem mass spectrometry (MS/MS). Gene ontology (GO) annotation of these proteins showed that most of the differentially expressed proteins were involved in response to stimulus and inflammatory response processes. Moreover, pulmonary surfactant-associated protein D and gelsolin, biomarkers of idiopathic pulmonary fibrosis, were significantly up-regulated in rat BALF after ZnO NPs exposure (2.42- and 2.84-fold, respectively). The results obtained from this present study could provide a complementary consequence with our previous study and contribute to a better understanding of the molecular mechanisms involved in ZnO NP-induced lung disorders.

Genetic Polymorphisms of SP-A, SP-B, and SP-D and Risk of Respiratory Distress Syndrome in Preterm Neonates

BackgroundWe examined selected polymorphisms in 3 pulmonary surfactant-associated proteins (SP) for their influence on serum SP levels and risk of respiratory distress syndrome (RDS) in preterm neonates.Material/MethodsPremature infants from a Han population were enrolled, including 100 premature infants with RDS (case group) and 120 premature infants without RDS (control group). SNP genotyping for SP-A (+186A/G and +655C/T), SP-B (−18A/C and 1580C/T), and SP-D (Met11ThrT/C and Ala160ThrG/A) used polymerase chain reaction-restriction fragment length polymorphism. Haplotypes were calculated with Shesis software and serum SP-A/B/D levels were quantified by ELISA.ResultsCase and control groups exhibited significant differences in genotype and allele frequencies of SP-A (+186A/G, +655C/T) and SP-B (1580C/T). However, no statistically significant differences were observed in the allele and genotype frequencies of SP-B −18A/C, SP-D Met11ThrT/C, and SP-D Ala160ThrG/A. Importantly, serum SP-A and SP-B levels were reduced in RDS patients carrying SP-A (+186A/G, +655C/T) and SP-B (1580C/T) polymorphisms. AA genotype of +186A/G, SP-A level, and CC genotype of 1580C/T were independently correlated with increased RDS risk.ConclusionsSP-A (+186A/G) and SP-B (1580C/T) polymorphisms are strongly associated with the risk of RDS in preterm infants. Notably, reduced serum SP-A levels were correlated with a high risk of RDS and may serve as novel biomarkers for RDS detection and monitoring.

Identification of vitamin D sensitive pathways during lung development.

BackgroundWe have previously shown that vitamin D deficiency has a detrimental impact on lung development. In this study, we aimed to identify the mechanisms linking vitamin D with lung development using a mouse model of dietary manipulation.MethodsFemale offspring were euthanized at different time-points; embryonic day (E)14.5, E17.5 or postnatal day (P)7. Lung tissue was collected for mass spectrometry-based proteomic analysis. Label-free quantitation was used to identify the differentially expressed proteins and ELISA confirmed the expression of selected proteins. Lungs from separate groups of mice were fixed and processed for stereological assessment of lung structure.ResultsNo differences in protein expression between vitamin D deficient and replete mice were detected at E14.5 and E17.5, whereas 66 proteins were differentially expressed in P7 lungs. The expression of pulmonary surfactant-associated protein B (SP-B) and peroxiredoxin 5 (PRDX5) were reduced in P7 lungs of vitamin D deficient mice, while the production of collagen type Ι alpha 1 (COL1A1) was higher in lungs of vitamin D deficient mice. There were no differences in lung volume, parenchymal volume, volume of airspaces or surface area of airspaces between vitamin D deficient and vitamin D replete mice across three time-points.ConclusionsThe difference in protein expression during the early postnatal time-point suggests that vitamin D deficiency may induce alterations of lung structure and function in later life during alveolarization stage through impaired pulmonary surfactant production and anti-oxidative stress ability as well as enhanced collagen synthesis. These data provided a plausible mechanism linking maternal vitamin D deficiency with altered postnatal lung function.

Effect research of two kinds of airway humidification solution for patients with tracheotomy of airway humidification

Objective To compare the influence of tracheotomy after two wet fluid on airway and provide the basis for clinical treatment and care. Methods A total of 30 patients with severe brain injury stay neurosurgery tracheotomy were divided into 0.45% sodium chloride group and ambroxol hydrochloride group with 15 cases each by random digits table method, two airway humidification liquid (0.45% sodium chloride, 0.9% sodium chloride + ambroxol hydrochloride)were each instilled in the trachea inner sleeve. Blood gas analysis was performed and the levels of serum lung surface active substances related protein-A (SP-A protein), interleukin-6, interleukin-8, tumor necrosis factor-alpha(TNF-α) were measured by enzyme linked immunosorbent assay (ELISA)before 1 d and after 3,7,14 d of tracheotomy. Results There were significant differences in arterial blood oxygen partial pressure, arterial carbon dioxide partial pressure, oxygenation index after 14 d of tracheotomy between ambroxol hydrochloride group and 0.45% sodium chloride group:(110.72±26.75) mmHg(1 mmHg=0.133 kPa) vs.(89.39±21.98) mmHg, (30.44±6.75) mmHg vs. (35.12±7.28) mmHg, 333.23±80.56 vs. 270.93±77.21,t=29.49,-8.63,7.44,P<0.01.There were significant differences in the levels of serum SP-A protein, interleukin -6, interleukin -8, TNF-α after 14 d of tracheotomy between ambroxol hydrochloride group and 0.45% sodium chloride group:(191.34±1.21) ng/L vs. (61.92±12.0) ng/L, (2.62±0.23) ng/L vs.(5.42±0.16) ng/L,(124.56±2.10) ng/L vs. (185.91±1.48) ng/L, (31.32±1.38) ng/L vs.(69.13±1.16) ng/L,t=75.72, -13.51, -23.89, -20.97,P<0.01. Conclusions The airway humidification effect of ambroxol hydrochloride group is better than 0.45% sodium chloride group, it can improve the wetting effect, and better protect the lung tissue, reduce the incidence of lung infection, make it an ideal airway humidification liquid. Key words: Craniocerebral trauma; Tracheotomy; Ambroxol; Pulmonary surfactant-associated protein A; Airway humidification

Invariant Natural Killer T Cells Play a Role in Chemotaxis, Complement Activation and Mucus Production in a Mouse Model of Airway Hyperreactivity and Inflammation.

CD1d-restricted invariant natural killer T (iNKT) cells play a critical role in the induction of airway hyperreactivity (AHR). After intranasal alpha-galactosylceramide (α-GalCer) administration, bronchoalveolar lavage fluid (BALF) proteins from mouse lung were resolved by two-dimensional differential gel electrophoresis (2D-DIGE), and identified by tandem mass spectroscopy. A lack of iNKT cells prevented the development of airway responses including AHR, neutrophilia and the production of the proinflammatory cytokines in lungs. Differentially abundant proteins in the BALF proteome of α-GalCer-treated wild type mice included lungkine (CXCL15), pulmonary surfactant-associated protein D (SFTPD), calcium-activated chloride channel regulator 1 (CLCA1), fragments of complement 3, chitinase 3-like proteins 1 (CH3LI) and 3 (CH3L3) and neutrophil gelatinase-associated lipocalin (NGAL). These proteins may contribute to iNKT regulated AHR via several mechanisms: altering leukocyte chemotaxis, increasing airway mucus production and possibly via complement activation.

Reproducibility of biomarkers in induced sputum and in serum from chronic smokers

RationaleSoluble inflammatory markers obtained from non-invasive airway sampling such as induced sputum may be useful biomarkers for targeted pharmaceutical interventions. However, before these soluble markers can be used as potential targets, their variability and reproducibility need to be established in distinct study populations. ObjectiveThis study aimed to assess the reproducibility of biomarkers obtained from induced sputum and serum in chronic smokers and non-smokers. MethodSputum and serum samples were obtained from 16 healthy non-smokers and 16 asymptomatic chronic smokers (for both groups: 8M/8F, 30–52 years, FEV1 ≥80% pred.; ≥10 pack years for the smokers) on 2 separate visits 4–10 days apart. Soluble markers in serum and sputum were analysed by ELISA. The differences between smokers vs non-smokers were analysed with a t-test and variability was assessed on log-transformed data by a mixed model ANOVA. ResultsAnalysable sputum samples could be obtained from all 32 subjects. In both study populations neutrophils and macrophages were the predominant cell types. Serum Pulmonary Surfactant Associated Protein D had favourable reproducibility criteria for reliability ratio (0.99), intra-subject coefficient of variation (11.2%) and the Bland Altman limits of agreement. Furthermore, chronic smokers, compared to non-smokers, had significantly higher sputum concentrations of IL-8 (1094.6 pg/mL vs 460.8 pg/mL, p = 0.006)), and higher serum concentrations of Pulmonary Surfactant Associated Protein D (110.9 pg/mL vs 64.7 pg/mL, p = 0.019), and lower concentrations of Serum Amyloid A (1352.4 pg/mL vs 2297.5 pg/mL, p = 0.022). ConclusionSerum Pulmonary Surfactant Associated Protein D proved to be a biomarker that fulfilled the criteria for reproducibility in both study groups.

Angiotensin-Converting Enzyme 2 Attenuates Bleomycin-Induced Lung Fibrosis in Mice

Background: Local renin-angiotensin system (RAS) activation has been shown to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). It has been reported that angiotensin-converting enzyme 2 (ACE2) could inhibit RAS-mediated epithelial injury and fibrogenesis and that ACE2 deficiency could aggravate acute and chronic lung injury. Through research, it could be deduced that ACE2 could protect against pulmonary fibrosis as a therapeutic target. Methods: Time-course analysis of the pathological characteristics of bleomycin-induced lung fibrosis was undertaken in a mouse model, and the effect of exogenous ACE2 on lung fibrosis was studied. Immunohistchemistry (IHC) staining and western blot (WB) testing for AGT and ACE2 were performed to evaluate the regulation of local RAS. TUNEL staining was used to observe epithelial apoptosis. Leukocyte common antigen (LCA) and pulmonary surfactant-associated protein A (SP-A) IHC staining and WB testing were performed to assess the inflammatory response and epithelial regeneration. Masson's staining and a hydroxyproline assay were performed to examine collagen deposition. IHC staining and WB testing for TGF-β1 and α-SMA were performed to investigate the regulation of pro-fibrotic cytokines and the activation of fibroblasts. Results: Exogenous ACE2 attenuated bleomycin-induced lung fibrosis by reversing the reduction of local ACE2 and by suppressing the elevation of AGT. ACE2 decreased the apoptosis index and LCA levels and ameliorated the dynamic change in SP-A level, thus protecting against epithelial injury. Reductions of TGF-β1 and α-SMA were also found in ACE2-treated mice, indicating the inhibition of fibrogenesis. Conclusion: ACE2 attenuated bleomycin-induced lung fibrosis as an anti-inflammatory anti-apoptotic and anti-fibrotic agent, and it might be a promising therapeutic target for IPF.

Identification and examination of a novel 9-bp insert/deletion polymorphism on porcine SFTPA1 exon 2 associated with acute lung injury using an oleic acid-acute lung injury model.

The pulmonary surfactant-associated protein (SFTPA1, SP-A) gene has been studied as a candidate gene for lung disease resistance in humans and livestock. The objective of the present study was to identify polymorphisms of the porcine SFTPA1 gene coding region and its association with acute lung injury (ALI). Through DNA sequencing and the PCR-single-strand conformation polymorphism method, a novel 9-bp nucleotide insertion (+) or deletion (-) was detected on exon 2 of SFTPA1, which causes a change in three amino acids, namely, alanine (Ala), glycine (Gly) and proline (Pro). Individuals of three genotypes (-/-, +/- and +/+) were divided into equal groups from 60 Rongchang pigs that were genotyped. These pigs were selected for participation in the oleic acid (OA)-ALI model by 1-h and 3-h injections of OA, and there were equal numbers of pigs in the control and injection groups. The lung water content, a marker for acute lung injury, was measured in this study; there is a significant correlation between high lung water content and the presence of the 9-bp indel polymorphism (P < 0.01). The lung water content of the OA injection group was markedly higher than that of the control group and lung water content for the +/+ genotype was significantly higher than that of the others in the 1-h group (P < 0.01). No differences in the expression of the SFTPA1 gene were found among individuals with different SFTPA1 genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The individuals with the -/- genotype showed lower lung water content than the +/+ genotype pigs, which suggests that polymorphism could be a potential marker for lung disease-resistant pig breeding and that pig can be a potential animal model for human lung disease resistance in future studies.

Effects of antenatal dexamethasone and ambroxol on expression of surfactant protein-B mRNA and ;thyroid transcription factor-1 in pulmonary tissue of premature rats

To explore the effects and mechanism of dexamethasone and ambroxol on expression of surfactant protein （SP）-B mRNA and thyroid transcription factor （TTF）-1 in premature rat lung. Methods Sixteen pregnant Sprague-Dawley rats were randomly divided into four equal groups： two doses of dexamethasone （0.2 mg/kg injected intramuscularly on Day 17 and 18 of pregnancy respectively）;single dose of dexamethasone （0.2 mg/kg injected intramuscularly on Day 18 of pregnancy）;ambroxol group （100 mg/kg injected intraperitoneally on Day 16, 17 and 18 of pregnancy respectively）; and control group （normal saline injected intraperitoneally on Day 16, 17 and 18 of pregnancy respectively）. There were four pregnant rats in each group. All of the fetal rats were taken out on Day 19 of pregnancy as the preterm birth model, and 20 fetal rats from each group were randomly selected. The ratio of body weight to fetal lung weight of newborn rats was calculated. Changes in lung morphology were observed under light microscopy and the ratio of alveoli surface area to alveolar septae surface area was calculated. Expression of TTF-1 protein was determined by immunohistochemistry. Expression of SP-B mRNA was detected by reverse transcriptase-polymerase chain reaction. One-way analysis of variance, Student-Newman-Keuls method and Pearson correlation analysis were applied as statistical methods. Results （1） The ratio of body weight to fetal lung weight was （6.5±0.6）, （7.9±0.8）, （9.5±0.8） and （9.5±0.9） mg/g in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=67.69,P〈0.01）. The ratio of two doses and one dose of dexamethasone group was lower than that of control group （q=17.143 and 9.143, all P〈0.01） and ambroxol group （q=17.143 and 9.143, all P〈0.01）. The ratio of two doses dexamethasone group was lower than that of one dose dexamethasone group （q=8.000, P〈0.01）. （2） The ratio of alveoli surface area to alveolar septae surface area was 2.19±0.15, 1.70±0.18, 1.67±0.13 and 1.08±0.12 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=190.85, P〈0.01）. The ratio of two doses of dexamethasone group, one dose of dexamethasone group and ambroxol group were higher than that of the control group （q=33.639, 18.788 and 17.879, all P〈0.01）. The ratio of two doses dexamethasone group was higher than that of one dose dexamethasone group （q=14.848, P〈0.01）. （3） Expression of TTF-1 protein was 0.311±0.018, 0.224±0.019, 0.196±0.013 and 0.191±0.018 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=211.69,P〈0.01）. TTF-1 protein expression of two doses and one dose of dexamethasone group were higher than that of control group （q=30.000 and 8.250, all P〈0.01） and ambroxol group （q=28.750 and 7.000, all P〈0.01）. TTF-1 protein expression of two doses dexamethasone group was higher than that of one dose dexamethasone group （q=21.750, P〈0.01）. （4） Expression of SP-B mRNA was 1.25±0.13, 1.15±0.12, 1.10±0.10 and 1.01±0.12 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=14.48, P〈0.01）. SP-B mRNA expression of two doses of dexamethasone group, one dose of dexamethasone group and ambroxol group were higher than that of control group （q=9.231, 5.385 and 3.462, all P〈0.01）. SP-B mRNA expression of two doses of dexamethasone group was higher than that of ambroxol group （q=5.769, P〈0.01） and one dose of dexamethasone group （q=3.846, P〈0.01）. （5）TTF-1 expression in two doses of dexamethasone group, one dose of dexamethasone group and control groups was positively correlated with SP-B mRNA expression （r=0.512, 0.597 and 0.449, respectively, all P〈0.05）. Conclusions Ambroxol can accelerate the maturation of fetal lung with minimal adverse effects on fetal lung weight. Ambroxol might be an alternative to dexamethasone to prevent neonatal respiratory distress syndrome. Key words: Dexamethasone; Ambroxol; Pulmonary surfactant-associated protein B; Nuclear proteins; Transcription factors; Infant,premature

Establishment of surfactant-associated protein A suicide gene system and analysis of its activity.

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.

Joint serum tumor markers serve as survival predictive model of erlotinib in the treatment of recurrent non-small cell lung cancer

背景与目的分子靶向治疗是肺癌个体化治疗的方向，目前已有学者建立靶向治疗预测模型，为临床个体化治疗提供更多的指导。本研究探讨血清肺表面活性物质相关蛋白（pulmonary surfactant-associated protein D, SP-D）、转化生长因子-α（transforming growthfactor α, TGF-α）、基质金属蛋白-9（matrix metalloproteinase 9, MMP-9）、组织多肽特异性抗原（tissue polypeptide specific antigen, TPS）、肺腺癌相关抗原（Krebs von den Lungen-6, KL-6）与晚期复治非小细胞肺癌（non-small cell lung cancer, NSCLC）治疗疗效及生存的关系，并构建生存预测模型。方法采用酶联免疫吸附法（enzyme-linked immuno sorbent assay, ELISA）检测114例晚期复治NSCLC患者治疗前外周血清中SP-D、TGF-α、MMP-9、TPS、KL-6含量，结合临床因素分析与厄洛替尼治疗疗效的关系，采用Kaplan-Meier生存曲线、Cox多因素生存分析模型进行单因素和多因素分析，并构建生存预测模型。结果114例患者厄洛替尼治疗总有效率为22.8%，稳定率为72.8%，中位无疾病进展时间（progression-free survival, PFS）为5.13个月，1年生存率为69.3%。SP-D > 110 ng/mL组的有效率及稳定率均高于≤110 ng/mL组（P=0.011, P=0.017），MMP-9≤535 ng/mL的稳定率高于 > 535 ng/mL组（P=0.009）。TPS < 80 U/L组的稳定率要高于≥80 U/L组（P=0.002）。SP-D > 110 ng/mL组的mPFS长于≤110 ng/mL组（5.95个月vs 3.25个月，P=0.009），MMP-9≤535 ng/mL的mPFS要长于 > 535 ng/mL组（5.83个月vs 3.47个月，P=0.046），KL-6中 < 500 U/mL组要优于≥500 U/mL组（6.03个月vs 3.40个月，P=0.040），TPS < 80 U/L组的mPFS要长于≥80 U/L组（6.15个月vs 2.42个月，P=0.014）。多因素分析显示吸烟史、EGFR基因野生型、末次化疗疗效进展、厄洛替尼治疗期间无皮疹、LDH升高和TPS≥80 U/L是PFS不佳的独立影响因素。通过建立预后预测模型，根据患者的预后指数可分成4组：低危组、中低危组、中危组和高危组，中位PFS分别是9.12个月、6.88个月、3.52个月和0.93个月，组间差异具有统计学意义（P < 0.001）。结论血清肿瘤标志物TPS水平联合患者临床特征建立预测厄洛替尼治疗晚期复治NSCLC生存模型，在临床上有一定的指导意义。

Role of cAMP-PKA signal transduction pathway in lidocaine-induced up-regulation of expression of surfactant protein-A in rat alveolar epithelial type II cells

Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal transduction pathway in lidocaine-induced up-regulation of the expression of surfactant protein-A (SP-A) in rat alveolar epithelial type Ⅱ cells (AEC Ⅱ).Methods Healthy male Sprague-Dawley rats were sacrificed and AEC Ⅱ were isolated,purified and incubated in 24-well culture plates (100μd/hole) with density of 1 × 106/ml.After being incubated for 2 h,the culture medium was replaced with serum-free medium DMEM.The cells were randomly divided into 4 groups (n =48 each):control group (group C),forskolin (adenylate cyclase agonist) group (group F),lidocaine 200 μg/ml group (group L),and PKA inhibitor H89 + L group (group P+ L).Forskolin 10 μmol/ml was added to DMEM in group F.Lidocaine 200 μg/ml was added to DMEM in group L.H89 10μnol/ ml was added to DMEM and AEC Ⅱ were incubated for 10 min,and then lidocaine 200 μg/ml was added in group P + L.At 6,12 and 24 h of incubation (T1-3),cAMP content and PKA activity (using ELISA),and expression of SP-A mRNA (by real-time fluorescent quantitative PCR) and SP-A (by Western blot) were measured.Results Compared with group C,the expression of SP-A mRNA and SP-A was significantly upregulated,and cAMP level and PKA activity were increased at T1-3 in group F and at T2,3 in group L.Compared with group L,the expression of SP-A and SP-A mRNA was down-regulated,PKA activity was decreased,and no significant change was found in cAMP level at T1-3 in group P + L.Conclusion Lidocaine can up-regulate the expression of SP-A in AEC Ⅱ of rats through activating cAMP-PKA signal transduction pathway. Key words: Lidocaine; Cyclic AMP; Cyclic AMP-dependent protein kinases; Epithelial cells; Pulmonary alveoli; Pulmonary surfactant-associated protein A

Observation of the effect of pulmonary surfactant-associated proteins combined with ambroxol in the treat-ment of 180 children with congenital alveolar dysplasia neonatal respiratory distress syndrome

Objective To observe the effect of pulmonary surfactant-associated proteins （ Kelisu） combined with ambroxol in the treatment of 180 congenital alveolar dysplasia children with neonatal respiratory distress syndrome （ NRDS） .Methods According to random number table ,180 congenital alveolar dysplasia children with NRDS were randomly divided into the observation group and control group , 90 cases in each group .In addition to conventional therapy,the control group was given ambroxol hydrochloride treatment ,the observation group was given Kelisu on thebasis of treatment of the control group.The treatment effect was compared between the two groups .Results Comparedwith the control group,after treatment the values of PaO2 and pH were significantly increased,PaCO2 significantly decreasedin the observation group（t =5.91,13.16,29.89,2.98,5.11,8.13,all P 〈0.05）.The duration of mechanicalventilation,length of hospital stay were （45.30 ±5.35）h,（10.01 ±2.24）d in the observation group,respectively,which were significantly better than[（68.27 ±6.13）h,（17.24 ±2.39）d]in the control group（t =26.78,20.94,all P 〈0.05）.Conclusion Kelisu combined with hydrochloride ambroxol can significantly improve hypoxia symp -toms in NRDS children,reduce the duration of mechanical ventilation and length of hospital stay ,improve the prognosis. Key words: Respiratory distress syndrome,newborn; Pulmonary surfactant-associated proteins; Ambroxol

Effect of hypoxia on lung gene expression and proteomic profile: Insights into the pulmonary surfactant response

Exposure of lung to hypoxia has been previously reported to be associated with significant alterations in the protein content of bronchoalveolar lavage (BAL) and lung tissue. In the present work we have used a proteomic approach to describe the changes in protein complement induced by moderate long-term hypoxia (rats exposed to 10% O2 for 72h) in BAL and lung tissue, with a special focus on the proteins associated with pulmonary surfactant, which could indicate adaptation of this system to limited oxygen availability. The analysis of the general proteomic profile indicates a hypoxia-induced increase in proteins associated with inflammation both in lavage and lung tissue. Analysis at mRNA and protein levels revealed no significant changes induced by hypoxia on the content in surfactant proteins or their apparent oligomeric state. In contrast, we detected a hypoxia-induced significant increase in the expression and accumulation of hemoglobin in lung tissue, at both mRNA and protein levels, as well as an accumulation of hemoglobin both in BAL and associated with surface-active membranes of the pulmonary surfactant complex. Evaluation of pulmonary surfactant surface activity from hypoxic rats showed no alterations in its spreading ability, ruling out inhibition by increased levels of serum or inflammatory proteins. This work reveals that hypoxia induces extensive changes in the proteomic profile of lung bronchoalveolar lavage, including the presence of proteins related with inflammation both in lung tissue and lavage, and a significant increase in the synthesis and secretion by the lung tissue of different forms of hemoglobin. The level of specific pulmonary surfactant-associated proteins is not substantially altered due to hypoxia, but hypoxia-adapted surfactant exhibits an enhanced ability to form surface-active films at the air-liquid interface. The increased amount of β-globin integrated into the operative surfactant complexes obtained from hypoxic rats is a relevant feature that points to the existence of adaptive responses coupling surfactant function and oxygen availability.