Mutual regulation between miR-21 and the TGFß/Smad signaling pathway in human bronchial fibroblasts promotes airway remodeling.

OBJECTIVE: Airway remodeling is an important pathological feature of asthma. Excessive deposition of extracellular matrix (e.g., collagen) secreted from fibroblasts is a major factor contributing to airway remodeling. Currently, the mechanism by which collagen continues to be oversynthesized in the airway remains unclear. In this study, we investigated the role of the microRNA-21 (miR-21) and TGFß/Smad signaling pathway in human bronchial fibroblasts (HBFs), and explored the regulatory mechanism of airway remodeling. METHODS: HBFs were cultured in vitro and treated with the transforming growth factor ß (TGFß), receptor inhibitor (SB431542), and TGFß1. miR-21 and Smad7 overexpressing lentiviruses, as well as an miR-21 interfering lentivirus were constructed and transfected into HBFs. Western blotting was used to determine the expression of airway remodeling-related proteins and proteins in the TGFß/Smad signaling pathway. miR-21 expression was measured by quantitative real-time PCR. RESULTS: The high expression of miR-21 induced by TGFß1 was reduced following the treatment with the SB431542 in HBFs. Smad7 overexpression inhibited the elevated expression of the COL I protein induced by miR-21 overexpression in HBFs. Inhibiting miR-21 expression upregulated the level of Smad7 protein, thus reducing the expression of airway remodeling-related proteins induced by TGFß1 stimulation in HBFs. CONCLUSIONS: TGFß1 can induce miR-21 expression in HBFs through the TGFß/Smad signaling pathway to promote airway remodeling. miR-21 downregulates Smad7, activates the TGFß/Smad signaling pathway, and promotes airway remodeling. Mutual regulation between miR-21 and the TGFß/Smad signaling pathway in HBFs promotes airway remodeling.

[Association between suppressors of cytokine signaling mRNA expression and Th1/Th2 balance in children with asthma].

OBJECTIVE: Suppressors of cytokine signaling (SOCS) have been shown to play an important role in regulating cytokines, such as intracellular interferon (IFN) and interleukin (IL), in the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. At present, the association between SOCS and asthma is still under study. The aim of this study is to explore the relationship of SOCS-1 and SOCS-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) with the intracellular IFN-'/IL-4 ratio in CD4+ T cells and specific IgE (sIgE) level in children with asthma. METHODS: BMCs were collected from 44 children with allergic asthma (4-14 years) and 30 healthy children. The intracellular IFN-'/IL-4 ratio in CD4+ T cells was measured by flow cytometry. Total RNAs were extracted from the PBMCs and SOCS-1 and SOCS-3 mRNA expression was measured by SYBR Green I quantitative RT-PCR. RESULTS: Compared with the healthy children, children with allergic asthma showed a lower level of intracellular IFN-' in peripheral blood [(15.7±2.0)% vs (19.1±2.7)%] and IFN-'/IL-4 ratio (3.4±1.5 vs 4.8±2.9) and higher SOCS-1 mRNA expression (-Ct, 11.1±1.9 vs 12.6±2.8). There was a negative relationship between SOCS-1 mRNA expression and the percentage of IFN-'-producing CD4+ T cells in peripheral blood in both asthmatic and healthy children (P<0.05). No correlation was found between SOCS-1 and SOCS-3 expression and sIgE level. CONCLUSIONS: Children with allergic asthma have elevated levels of SOCS-1 mRNA in PBMCs, which is associated with Th2-skewed immune response.

[Roles of allergen testing in vitro and impulse oscillometry for lung function measurements in children with cough variant asthma].

OBJECTIVE: To study the roles of allergen testing in vitro and impulse oscillometry for lung function measurements in preschool children with cough variant asthma (CVA). METHODS: ethodsForty-four preschool children with acute asthma, 41 with chronic asthma, 46 with CVA, and 35 healthy preschool children as control were recruited in the study. Inhaled allergen, food allergen, and mite-specific IgE were determined by Pharmacia UniCAP System. Serum eosinophil cationic protein (ECP) and total IgE levels were measured. Lung function was assessed by impulse oscillometry. RESULTS: The positive rates of inhaled allergen and food allergen, and total IgE levels in the CVA, acute asthma and chronic asthma groups were higher than those in the control group (P<0.01). However, no significant differences were found among the three case groups. The serum ECP levels in the CVA group were lower than those in the acute asthma group (P<0.01), but did not show differences when compared with the chronic asthma group. The impulse oscillometry demonstrated that the respiratory total impedance (Zrs), airway resistance at 5 Hz (R5), airway resistance at 20 Hz (R20), subtracting R5 from R20 (R5-R20) and resonant frequency (Fres) in the CVA, acute asthma and chronic asthma groups were higher than those in the control group (P<0.01). Zrs, R5, R20, R5-R20, and Fres in the CVA and chronic asthma groups were lower than those in the acute asthma group (P<0.01). Serum ECP levels were positively correlated with Zrs, R5, R5-R20 and Fres (P<0.05) in the CVA and chronic asthma groups. CONCLUSIONS: The measurements of allergens, serum ECP and impulse oscillometry for lung function are helpful for the evaluation of airway inflammation and airway obstruction in preschool children with CVA.

[Differentiation of QY1 bone marrow pluripotential mesenchymal stem cell line cells into cardiomyocytes and vascular endothelial cells in vitro].

OBJECTIVE: To explore the differentiation potential of QY1 bone marrow mesenchymal stem cell (MSCs) line cells into cardiacmyocytes and vascular endothelial cells in vitro, to optimize the suitable conditions of MSCs differentiating into cardiomyocytes in vitro, and to examine the potentials of MSCs differentiating into cardiomyogenesis and vasculogenesis. METHODS: Specifically committed differentiation inductive medium was employed, including 5-azacytidine for cardiomyogenesis and vascular endothelial growth factor for vasculogenesis in culture respectively in vitro. The differentiated cells were identified by immunohistochemistry and molecular biology. RESULTS: MSCs line cells had been cultured in the normal culture medium for 72 hours, then the differentiation inductive medium including 10 micromol/L 5-azacytidine was added into the normal culture dishes for 24 hours only. After that the culture medium was changed back to the normal culture medium. Normal culture medium was changed every 7 days. The second induction was performed after 14 days. The differentiated cells treated with 5-azacytidine could beat spontaneously and formed myotube structures in the optimal induction conditions, and the differentiation rate was (39.47+/-0.56)%. The differentiated cells expressed specific cardiomyocytic proteins identified by the positive immunohistochemistry staining with anti-alpha-sarcomeric antibody and anti-Cx-43 antibody, and also expressed the alpha-myosin heavy chain examined by RT-PCR. The differentiated cells began to appear as the lined up vascular endothelial cells after 48 hour treatment with vascular endothelial growth factor. Some of the differentiated cells connected each other to form vascular endothelial web-like structure after 7 day treatment with vascular endothelial growth factor. On 14 d after treating with vascular endothelial growth factor, the differentiated cells were identified by immunohistochemistry staining. The expressions of both specific surface antibody CD31 and factor VIII for vascular endothelial cells were positive. CONCLUSION: The cells of QY1 bone marrow mesenchymal stem cell line may differentiate into cardiomyocytes or vascular endothelial cells in vitro under specific condition.

[Pluripotential differentiation of QY1 bone marrow mesenchymal stem cell line].

OBJECTIVE: To explore the ability of QY1 bone marrow mesenchymal stem cell (MSCs) line cells to differentiate into adipocytes, chondrocytes, osteoblasts, cardiac myocytes,vascular endothelial cells, and neural cells in vitro. METHODS: The QY1 cells at passage 5 were treated with the adipogenic medium, the chondrogenic medium and the osteogenic medium, 5-azacytidine, vascular endothelial growth factor and neural cell medium (revulsant 1 was 10 mmol/L beta-mercaptoethanol; revulsant 2 was 2%dimethylsulfoxide and 10(-8)mol/L dexamethasone) in culture respectively in vitro. The differentiated cells were identified by staining, immunohistochemistry and RT-PCR. RESULTS: The differentiated cells induced by the adipogenic medium formed adipocytes and contained fat lipid droplets, which were stained positively with Sudan III after 21 days of culture. The differentiated cells induced by the chondrogenic medium formed chondrogenic nodules, which were stained positively by Alcian blue at pH 1.0 after 21 days of culture. The differentiated cells induced by the osteogenic medium formed osteogenic nodules, which were stained positively by Von Kossa staining after 35 days of culture, and the secretion of a calcified extracellular matrix as black nodules was observed. The differentiated cells treated with 10 micromol/L 5-azacytidine could beat spontaneously and formed myotube structures,which were identified by the positive immunohistochemistry staining with anti-alpha-sarcomeric antibody and anti-Cx-43 antibody. The expression of alpha-myosin heavy chain was also observed by RT-PCR. The differentiated cells treated with 50 ng/mL vascular endothelial growth factor could form vascular endothelial cells and vascular endothelial web like structure, which were identified by the positive immunohistochemistry staining with CD31 and Factor VIII. The differentiated cells induced by revulsant 1 were positive in the immunohistochemistry staining with neuron-specific nuclear protein, while the expression of glial fibrillary acidic protein was negative. The differentiated cells induced by revulsant 2 were positive in the immunohistochemistry staining with glial fibrillary acidic protein, while the expression of neuron-specific nuclear protein was negative. CONCLUSION: QY1 bone marrow mesenchymal stem cell line has the ability to differentiate into adipocytes, chondrocytes, osteocytes, cardiomyocytes, vascular endothelial cells, neurons and neural glial cells in vitro. A bone marrow mesenchymal stem cell line cell can at least differentiate into 7 types of cells, which come from mesoderm and ectoderm.