In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52±0.05) was lower than that of the N group (0.72±0.06) (P〈0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522±0.0512) was significantly lower than that of the N group (0.4432± 0.05628) (P〈0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.
Background The decrease of surfactant protein (SP) secreted by the alveolar type Ⅱ cell is one of the important causes of limiting air of pulmonary emphysema. However, the SP-A gene and protein changes in this disease are rarely studied. This study was undertaken to investigate alterations in SP-A gene activity and protein, and to explore their roles in the pathogenesis of emphysematous changes. Methods Twenty Wistar rats were divided randomly into a normal control group (n=10) and a cigarette smoking (CS) + lipopolysaccharide (LPS) group (n=10). Ultra-structural changes were observed under an electron microscope. The number of cells positive for SP-A was measured by immunohistochemistry. The mRNA expression and protein level of SP-A in the lung tissues were determined by quantitative polymerase chain reaction (qPCR) and Western blot separately. The protein level of SP-A in lavage fluid was determined by Western blot. Results The number of cells positive for SP-A of the CS+LPS group (0.35±0.03) was lower than that of the blank control group (0.72±0.06, P 〈0.05). The level of SP-A in the lung tissues of rats in the CS+LPS group (0.2765±0.0890) was lower than that in the blank control group (0.6875±0.1578, P 〈0.05). The level of SP-A in the lavage fluid of rats in the CS+LPS group (0.8567±0.1458) was lower than that in the blank control group (1.3541±0.2475, P 〈0.05). The lung tissues of rats in the CS+LPS group showed an approximate increase (0.4-fold) in SP-A mRNA levels relative to β-actin mRNA (P 〈0.05). Conclusions The changes of SP-A may be related to emphysematous changes in the lung. And cigarette smoke and LPS alter lung SP-A gene activity and protein homeostasis.
HU Qiong-jie XIONG Sheng-dao ZHANG Hui-lan SHI Xue-mei XU Yong-jian ZHANG Zhen-xiang ZHEN Guo-hua ZHAO Jian-ping
In order to investigate the effect of interleukin-18 (IL-18) on airway inflammation in asthmatic murine models and its mechanisms, BALB/C mice were randomly divided into three groups (n=10 in each group): group A (control group); group B (asthmatic model group); group C (IL-18-treated group). The asthmatic model was established in groups B and C by respiratory syncytial virus (RSV) killed by ultraviolet. Saline solution (0.1 mL) and IL-18 (0.1 mL, 1μg) were intraperitoneally injected respectively in groups B and C at 7 time points (day 1, 2, 7, 8, 9, 21, 22). The number of eosinophils (EOS) and plasmacytes in the airway was observed. The levels of interferon gamma (IFN-γ) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The results showed that symptoms of asthma in group C were more severe than in groups A and B. In group A, there were no EOS and plasmacytes in the airway submucosa. The number of EOS [ 15±3 (average cell counts per microscopic visual field, the same below)] and plasmacytes (10±2) in group B were increased significantly. However, the number of EOS and plasmacytes in group C (6±2 and 2±1, respectively) was decreased significantly as compared with group B (both P〈0.05). The levels of IFN-γ in groups A, B and C were 31±3, 40±5 and 63±5 μg/mL respectively, and those in group C were significantly higher than in groups A and B (both p〈0.05). It was suggested that the mechanism by which IL-18 inhibited the airway inflammation in asthmatic mice might be contributed to the fact that IL-18 could induce the induction of IFN-γ.
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 trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin (i The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression 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 purification by using IgG, with a yield of about 2-3 × 10^7, 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.
Summary: 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 ceils 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.
Summary: To study the role and mechanisms of hypoxia-inducible factor-lalpha (HIF-1α on the growth and tumorigenicity of lung cancer cells A549, the antisense oligonucleotide of HIF-1α was transfected to A549 cells. The effect of the antisense oligonucleotide on tumor growth in vitro and in vivo was evaluated by the growth rate suppression of A549 cells and subcutaneous implanted tumor in nude mice, and the effect on tumorigenicity was evaluated by the expression inhibition of angiogenic factors, the microvessel density (MVD)and vascular endothelial growth factor (VEGF) protein expression which were detected by immohistochemistry and western blot respectively. This study revealed that in vitro the growth rate of antisense oligonucleotide group was significantly decreased as compared with that of control group, sense oligonucleotide group and false-sense oligonucleotide group; in vivo the weight of implanted tumors in nude mice of antisense oligonucleotide group was 1.51±0.40 g, which was significantly lower than that of control group (2.79±0.33 g), sense oligonucleotide group (2.81±0.45g) and false-sense oligonucleotide group (2.89±0.39 g) and the inhibitory rate was 47 %. Both MVD and VEGF protein expression were significantly inhibited in antisense oligonucleotide group compared with those in other groups. These results indicated that antisense oligonucleotide of HIF-1α could inhibit lung cancer cells A549 growth in vitro and in vivo, and the mechanism may be due to the inhibition of vascular growth and VEGF protein expression.
The effect and mechanism of the ciglitazone on lung cancer cells A549 growth in vitro and in vivo were studied. Various concentrations of ciglitazone were added to the cultured A549 line, and the proliferation and differentiation of A549 cells were examined by MTT and cytometry analysis. A549 cells (1 × 10^6/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups., the control group, the ciglitazone treated group. The weights of subcutaneous tumors were measured, The expression of cyclin D1 and P21 in the lung was detected by immohistochemistry and Western blot respectively. The results showed that the proliferation of A549 was inhibited significantly by ciglitazone in a dose- and time-dependent manner. There were more ceils arrested in G1/G0 phase and the expression of PPARγ was markedly upregulated in ciglitazone-treated group. Direct injection of ciglitazone into A549-induced tumors could suppress tumor growth in nude mice and the growth inhibitory rate was 36 %. The expression of cyclin D1 was decreased and P21 increased significantly in ciglitazone-treated group as compared with control group. It was concluded that ciglitazone could inhibit A549 proliferation dose-dependently and time-dependently and induce differentiation, ,which might be related to the modulation of cell cycle interfered by PPARγ.
Background Expression of murine calcium-activated chloride channel family member 3 (mCLCA3) has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin (OVA). However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified. Methods mCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid (BALF) were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC (MUC5AC) were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 (IL-13) were determined quantitatively. Results mCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-y, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group (P 〈0.05). The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALE The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue. Conclusion These findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may i
MEI LiHE LiWU Si-siZHANG BoXUYong-jianZHANG Zhen-xiangZHAO Jian-pingZHANG Hui-lan