ABSTRACT
Objective To explore the influence and mechanism of cytokines and protein expression of alveolar epithelial type (ACE) Ⅱ cells in Bama minipigs' right-thorax with a single 15 Gy dose irradiation.Methods All minipigs received either right thoracic irradiation or sham-irradiation under anesthesia.At 4,8,12 and 24 week post-irradiation,5 minipigs respective and random from irradiarion groups and control group were sacrificed to remove the lungs.The protein expression of surfactant associated protein (SP)-A,transforming growth factor (TGF)-β1,Vimentin and E-cadherin were detected by Western blot.The protein expression of α-smooth muscle actin (SMA) was detected by immunohistochemistry.The co-localization of SP-A and α-SMA was visualized by double immunofluorescence staining.Results At 4,8,12 and 24 week post-irradiation,a significant increase in the protein expression of α-SMA,TGF-β1 and Vimentin were observed in irradiated lung compared to sham-irradiated controls(α-SMA:t =2.46-3.26,P ＜0.05;TGF-β1:t =2.96-3.52,P ＜0.05;Vimentin:t =3.24-5.05,P ＜ 0.05).By contrast,the protein expression of SP-A and E-cadherin in irradiation group was lower than it in control group (SP-A:t =3.62-4.65,P ＜ 0.05;E-cadherin:t =2.53-4.15,P ＜ 0.05).Moreover,at 8 week after irradiation,under confocal laser scanning microscope,the co-localization of SP-A and α-SMA was observed in irradiated alveolar epithelium cells,and it was not observed in sham-irradiated controls.Conclusions These data demonstrate that E-cadherin,SP-A and TGF-β1 may act as sensitive predictors of radiation-induced lung injury(RILI).Irradiation may lead to ACE Ⅱ cells achieving a mesenchymal phenotype,namely,epithelial to mesenchymal cells transition occurs,and ACE Ⅱ cells play the important part in the development of RILI by epithelial-mesenchymal transition.
ABSTRACT
Objective To explore the survival/proliferation,apoptotic and death effects of keratinocyte growth factor (KGF) in alveolar epithelial type Ⅱ cells (AT Ⅱ Cs) exposed to hyperoxia.Methods Primary culture of AT Ⅱ Cs from the Sprague-Dawley rat fetuses was studied under room air condition (210 mL/L O2) and hyperoxic condition (950 mL/L O2) for 0.5-12.0 h.Various concentrations of KGF (15 μg/L,25 μg/L,50 μg/L,75 μg/L,100 μg/L)were added into the cell cultures.Cells were randomly divided into room-air group,room-air-KGF group,hyperoxic-exposure group and hyperoxic-exposure-KGF group.The levels of intracellular reactive oxygen species (ROS),cleaved cysteinyl aspartate specific proteinase-3 (Caspase-3),cell death and proliferation of AT Ⅱ Cs were measured by flow cytometer,Western Blot,release of lactate dehydrogenase assays (LDH assays) and 3-(4,5-Dimethyhhiazol-2-yl)-2,5-diphenyhetrazolium bromide assays (MTT assays),respectively.Results Under room air condition,KGF could significantly increase AT Ⅱ Cs proliferation with 15-100 μg/L in a dose-dependent manner and significantly decrease LDH production at concentrations of 25-100 μg/L.Exposure to hyperoxia resulted in a significant increase in intracellular ROS production in AT Ⅱ Cs in a time-dependent manner compared with that of the room air group.Cell viability decreased and LDH release increased significantly in a time-dependent manner when AT Ⅱ Cs were exposed to 950 mL/L O2 for more than 4 h.After exposure to hyperoxia for 0.5 h and 1 h,KGF could significantly increase AT Ⅱ Cs proliferation in 15-75 μ g/L and significantly decrease LDH production at concentrations of 25-75 μg/L.After exposure to hyperoxia up to 4 h,higher viability was observed in 15 μg/L and 25 μg/L KGF group,and lower death rate presented in 25-100 μg/L KGF group.Further,prolnged hyperoxic exposure for 8 h,high viabilitv was shown only in 50 μg/L KGF group,and less death rate was observed only in 75 μg/L KGF group.In addition,no significant difference in viability and mortality was found between hyperoxic group and hyperoxic-KGF group after hyperoxic exposure for 12 h.Expression of cleaved Caspase-3 was significant higher after 4 h and 8 h hyperoxic exposure than that in room-air group ;at the same time,by adding 25 μg/L and 75.μg/L KGF led to decreased expression of Caspase-3 was detected,compared to hyperoxic group.Conclusions KGF may promote survival/proliferation,inhibited apoptosis and death of rat fetal AT Ⅱ Cs in room air condition or under temporary exposure to hyperoxia in vitro.However,prolonged exposure to hyperoxia may decrease the sensitivity of AEC Ⅱ Cs to KGF and limit its protective effects on lung injury.
ABSTRACT
Objective To investigate the effect of hyperoxia on the expression and transport function of epithelial sodium channel (ENaC) in neonatal rat alveolar epithelial type Ⅱ(AT Ⅱ) cells.Methods AT Ⅱ cells were isolated from neonatal rats,and primarily cultured under hyperoxic or normoxic conditions.Western blot was applied to examine the ENaC expression,and the amiloride-sensitive Na + currents were recorded using the whole-cell patch clamp technique.Results Hyperoxia upregulate the expression of β-ENaC and γ-ENaC subunits in the neonatal rat ATⅡ cells(β-ENaC:1 d:0.43 ±0.06 vs0.32 ±0.04,P =0.047;2 d:0.73±0.06 vs 0.50±0.08,P =0.019;3 d:0.72 ±0.08 vs 0.52 ±0.06,P =0.027;γ-ENaC:1 d:0.64±0.05 vs0.53 ±0.05,P =0.044;2 d:0.76 ±0.03 vs 0.52 ±0.04,P =0.001 ;3 d:0.77 ±0.06 vs 0.61 ±0.05,P =0.025).In addition,the amiloride-sensitive Na+ currents in hyperoxia-exposed AT Ⅱ cells were also increased (1d:13.71 ±2.77 vs8.92±1.38,P＜0.001;2d:29.12±11.03 vs 10.41 ±1.80,P＜0.001),which was consistent with the upregulated expression of β-ENaC and γ-ENaC.However,the expression of α-ENaC was inhibited by hyperoxia to some extent (1 d:0.31 ± 0.05 vs 0.46 ± 0.05,P =0.025 ; 2 d:0.30 ±0.01 vs0.38±0.02,P=0.002;3d:0.37±0.06 vs 0.37 ± 0.08,P =0.983).Conclusion Hyperoxia enhanced the transport function of ENaC in neonatal rat AT Ⅱ cells.Dysfunctional transport of Na + may not be a key factor involving in pulmonary edema at the early stage of bronchopulmonary dysplasia.
ABSTRACT
To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, which were then purified by plated into culture flask coated with rat immu- noglobulin G. The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi-croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expres-sion and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive pu- rification by using IgG, with a yield of about 2-3×107, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.