Between Bone Marrow Mesenchymal Stem Cells In Treating Bronchial Asthma With The Role Of Research

Objective1. To establish asthmatic mice model by sensitiziation/exposure with ovalbumin.2. To analyze histopathologic manifestation of lungs in the asthmatic mice and the cellural count in bronchial alveolar lavage fluid (BALF).MethodsTwenty BALB/c mice were divided into control group and asthma group equally. The mice in asthma group were sensitized with ovalbumin via twice intraperitoneal injections, on day0and14of the experiment. From day 24to26after the second sensitization, the mice were stimulated with aerosolized ovalbumin for30minutes per day. The control group were treated with normal saline. All mice were sacrificed48hours later after the last stimulation with overdose of ketamine in order to analyze the histological changes of the lung tissue and cellural count in BALF.Result1. Histopathological changes in mice after the sensitization and stimulat-ion with ovalbumin were correspondent to the typical manifestations of inflammation in asthma.2. Being compared with control group, the total cellural count was obviously increased in asthma group, especially for eosinophile granulocyte.3. The level of serum IL-4increased while INF-γ in BALF decreased in mice of asthma group.Conclusion1. We established asthmatic mice model successfully.2. We identified again that the disorders of asthma might due to the imbalance of Thl/Th2, and Th2cells and their cytokines played a vital role in the mechamism of asthma. Objective1. To isolate and culture mice bone marrow mesenchymal stem cells in vitro.2. To observe the morphology of BMSC and to identify the cell by flow cytomestry with the special markers.MethodsAfter the mouse was killed, their femurs and tibias were dissected away from attached muscle and connective tissues, and the ends of the bones were removed. Bone marrow was rushed out by normal saline and the red blood cells were eliminated with erythrocyte lysate.2×106marrow cells were placed in a75cm2tissue culture flask in DMEM cultural medium containing10%fetal bovine serum (FBS),1%penicillin and streptomycin at37℃in a humidified atmosphere of5%carbon dioxide. Cultural medium were changed every48hours. Cultured BMSCs were observed under inverted microscope, and the level of expansion was valued and the morphology was verified at each time when cultural medium were changed. In order to prevent the BMSCs from differentiation or decrease the rate of division, primary cultured cells was passaged when the cell density within colonies became80-90%confluent. The adherent BMSCs were released and collected from flasks with0.25%trypsin in1mmol/1sodium ethylenediaminetetraacetic acid. After the twice-passaged cells became nearly confluent, they were harvested and used for the experiments. BMSCs were identified by flow cytomestry with CD29. CD34and CD44.ResultFlow cytometry analysis demonstrated that there were significant expressions of BMSC specific antigens (CD29, CD44) and absence of hematopoietic marker antigen (CD34).ConclusionBMSCs were isolated and cultured successfully in vitro. ObjectiveTo indentify if the intraperitoneally injection of BMSCs could relieve the pulmonary inflammation in asthmatic mice, and play a role in the therapy of asthma, and to explore its potential mechanism.MethodsThe mice were divided into five groups:1. Normol control group:normal saline was used.2. Asthmatic control group:OVA sensitize and exposure.3. Asthma+BMSCs therapy group:1×107BMSCs were intraperitoneal injected into asthmatic mice.4. Asthma+BMSCs medium therapy group:BMSCs medium was intraperitoneal injected into asthmatic mice.5. Asthma+BMSC lysate therapy group:1×107BMSCs lysate was intraperitoneal injected into asthmatic mice.Result:1. Asthmatic mice models were establishmed successfully.2. The therapeutic results of BMSCs in asthmatic mice were encouraging.3. The therapeutic results of BMSCs medium or lysate were positive, and there was no difference between them.4. No side effects were observated.Conclusion1. BMSCs could be used to treat acute asthma without side effects in short-term.2. The therapeutic results of BMSCs medium or lysate were positive, but their effections were weaker than BMSCs. ObjectiveTo detect the functional changes of T lymphocyte after being co-cultured with BMSCs, and to explore the potential mechanism of BMSCs in the therapy of asthma.MethodsT lymphocytes were isolated from spleen of mice, and co-cultured with BMSCs for72hours, and then collected the medium and tested the level of IL-4、IL-17、IFN-γ in the supernatant. The expression of Lck in T lymphocytes was detected with Western bolt. There were six groups:1. Culture of T lymphocytes from normal mice.2. Culture of T lymphocyte from asthmatic mice.3. Co-culture of T lymphocyte of normal mice with BMSCs from normal mice.4. Co-culture of T lymphocyte from asthmatic mice with BMSCs from asthmatic mice.5. Co-culture of T lymphocyte from asthmatic mice with BMSCs from normal mice.6. Co-culture of T lymphocyte from asthmatic mice with BMSCs from normal mice in homogenate of lung tissues.Result1. There were no differences for the level of IL-4、IL-17、IFN-γ among different groups.2. The expression of Lck in T lymphocyte of group1and group4were obviously higher than other groups.Conclusion1. BMSCs could inhibit the expression of Lck in T lymphocyte and control its function.2. The function of BMSCs from asthmatic mice might be damaged and could not play roles of immune suppression.