Characterization of air-liquid interface culture of A549 alveolar epithelial cells

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.

Protective effects of dexmedetomidine on alveolar epithelial cells in sepsis mice / 第二军医大学学报

Objective To explore the role of dexmedetomidine (DEX) in the inflammatory response of alveolar epithelial cells in sepsis mice. Methods Male C57BL/6 mice were randomly divided into cecal ligation and puncture (CLP) group and CLP＋DEX group (n＝36). The mice in the CLP group were intraperitoneally treated with 1 mL sterile normal saline and the mice in the CLP＋DEX group were intraperitoneally injected with DEX (50 μg/kg) at 15 min before CLP. The survival rate of mice was recorded within 24 h after CLP. The serum and bronchoalveolar lavage fluid (BALF) were collected on 0, 6, 12, 24 h after CLP, and the levels of interleukin (IL)-6, IL-1β and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The mouse alveolar epithelial cell lines MLE12 were cultured in vitro, and were divided into lipopolysaccharide (LPS) group (1 μg/mL LPS) and LPS＋DEX group (1 μg/mL LPS＋0.2 μg/mL DEX). The levels of IL-6, IL-1β and TNF-α in the cell supernatants were measured by ELISA, and the phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) were determined by Western blotting on 6, 12 and 24 h of cell culture. Results Compared with the CLP group, the survival rate of mice was significantly higher in the CLP＋DEX group within 24 h after CLP (P0.05). The IL-6, IL-1β, and TNF-α levels of serum and BALF were significantly lower in the CLP＋DEX group than those in the CLP group (P0.05, P0.01). Compared with the LPS group, the levels of IL-6, IL-1β and TNF-α were significantly lower in the MLE12 cell supernatant of the LPS＋DEX group on 6, 12 and 24 h of cell culture (P0.05, P0.01). Western blotting results showed that the phosphorylation levels of ERK1/2 on 6, 12 and 24 h of cell culture and the phosphorylation levels of JNK on 6 and 12 h of cell culture were significantly lower in the LPS＋DEX group than those in the LPS group (P0.05, P0.01). Conclusion DEX can reduce the production of inflammatory cytokines in the serum and BALF of sepsis mice and increase the survival rate in sepsis mice, which may be related to the inhibition effect of DEX against activation of ERK1/2 and JNK signal pathways.

Construction of pEGFP-N1-TGF-β1 recombinant plasmid and transfected it into primary cultured AEC-Ⅱ of neonatal piglet using lipofectamine 2000 / 中华急诊医学杂志

Objective To construct pEGFP-N1-TGF-β1 recombinant plasmid and transfect it into primary cultured neonatal piglet type Ⅱ alveolar epithelium cell (AEC-Ⅱ) by using lipofectamine 2000,in order to provide basis of methodology for producing recombinant plasmids for transplantation of transfected AEC-Ⅱ into ALI/ARDS animal model lungs.Methods PCR primers were designed to amplify the human TGF-β1 cDNA fragment from plasmid.XhoI and EcoRI were used for double digesting the empty plasmid pEGFP-N1 and cDNA fragment of human TGF-β1.Then the products of double enzyme digestion by using T4 DNA ligase were connected and transformed into DH5α and cultured over night for 16 hours.The structure of recombinant plasmid was identified by using PCR and base sequencing to verify the correctness of pEGFP-N1-TGF-β1 recombinant plasmid.It was then transfected into primarily cultured AEC-Ⅱ by lipofectamine2000 mediated transfection and cultured for another 48 hour.Plasmid DNA (pEGFP-N1-TGF-β1 recombinant plasmid) and lipofectamine 2000 were added into serum-free DMEM respectively,then DNA suspension and Lipofectamine 2000 suspension were blended together and added into cells.After 24-48 hours later,the expression level of enhanced green fluorescent protein (EGFP) was evaluated under fluorescence microscope.Results The structure of vector was verified as pEGFP-N1-TGF-β1 recombinant plasmid by using PCR and base sequencing.Green fluorescence found in some cells showed that the pEGFP-N1-TGF-β1 recombinant plasmids had been successfully transfected into primary cultured AEC-Ⅱ,however,the transfection efficiency still need tobe further improved such as repeating the transfection procedure once again or using adenovirus mediated transfection method to improve the efficiceny and to transplant the cells into animal lungs eventually.Conclusions pEGFP-N1-TGF-β1 recombinant plasmid was successfully constructed and,for the first time,transfected into primarily cultured AEC-Ⅱ of newborn piglets.This established method should be useful for investigation of therapeutic effect and outcomes of lung with experimental acute lung injury.

Effects of different mechanical ventilation methods on morphology of type Ⅱ alveolar epithelium cell in newborn piglets with acute lung injury / 中华实用儿科临床杂志

Objective To investigate the effects of 3 different ventilation methods,including conventional mechanical ventilation(CMV),high-frequency oscillatory ventilation(HFOV) and partial liquid ventilation(PLV),on the morphology of type Ⅱ alveolar epithelium cell (ACE Ⅱ) of newborn piglet with acute lung injury (ALI).Methods Twenty-four less than 3-day-old newborn piglets were enrolled.After ALI was established with saline lavage (38 ℃,0.035 L/kg),newborn piglets were randomly assigned to 4 study groups:control group(n =6,no ventilation),CMV group(n =6),HFOV group(n =6),and PLV group(n =6,38 ℃,0.018 L/kg).Piglets in the 4 groups were sacrificed after being ventilated for 24 hours,and the number,area,density of fluorescence of ACE Ⅱ were detected.Results Through 24 hours mechanical ventilation,the numbers of ACE Ⅱ in CMV group,HFOV group and PLV group remained were 30 ± 5,52 ± 5,81 ± 7,respectively,while areas of fluorescence were 340.40 ± 47.50,329.69 ± 124.50,295.55 ± 109.30,respectively,and there were significant differences among the 3 groups,and the population mean of density of fluorescence had significant difference among the 3 groups.The number of ACE Ⅱ remains was lowest in CMV group compared with HFOV group(P =0.026) and PLV group (P =0.000),and the density of fluorescence of ACE Ⅱ was lowest in CMV group compared with HFOV group (P =0.001) and PLV group (P =0.002).Conclusions Different mechanical ventilations have various effects for ACE Ⅱ,and CMV is the most harmful mechanical ventilation on ACE Ⅱ,while PLV is the least harmful.

Divergent effects of Nitric oxide on airway epithelial cell activation

Nitric oxide (NO*) is a gaseous mediator synthesized by Nitric oxide sinthases. NO* is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO* in the synthesis of the chemok Nes Interleukin-8 and Monocyte Chemotactic Protein-1, and of Intercellular Adhesion Molecule-1 by human airway epithelial cells. normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. Neterleukin-8 (IL-8) and Monocyte Chemotactic Protein-1 (MCP-1) secretion and Intercellular Adhesion Molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RTI-PCR. Neterleukin-8 secretion was significantly reduced after 24h incubation with the NO* donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obta Ned with S-Nitroso-N-D,L-penicillam Ne and S-Nitroso-L-glutathione. Inhibition of endogenous NO* with the Nitric oxide synthase inhibitor N-Nitro-L-arg N Ne-methyl-esther caused an increase in IL-8 secretion by lypopolisaccharide- and cytok Ne-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in Monocyte Chemotactic Protein-1 secretion by both cell types. In contrast, Intercellular Adhesion Molecule-1 expression was upregulated by sodium NItroprusside. RTI-PCR results indícate that the modulation of protein levels was paralleled by modification in mRNA levels. NO* has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.

Beta agonist regulation of sodium transport in fetal lung epithelium: roles of cell volume, cytosolic chloride and protein tyrosine kinase

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.