| BackgroundAt the moment, the morbidity rate of bronchus asthma gradually rise at home and abroad. There are about100million people suffer from bronchus asthma in the world, so as asthma has been the main chronic diseases threatened seriously to people’s health, and its pathogenesis has not yet fully understood. TH1/TH2balance theory has been the core of the pathogenesis of bronchial asthma in recent years. Research shows that in addition to Th1cells and Th2, there is a subtype of T cells which function and cell factors different from Th1and Th2, called regulatory T cells (regulatory T-cell, Treg). The regulatory T cells mainly includes:the secretion of high level IL-10Trl, secretion TGF-β primarily TH3and CD8+, CD4+regulatory T cells. Among them, the CD4+regulatory T cells induced the suppression effect, and the CD8+regulatory T cells play inhibition effect. CD8+CD28-regulatory T cells is one of the main subgroup which around90%accounted. It can inhibit the up-regulation of antigen presenting cells stimulating molecules of CD80、 CD86、CD54、CD58. In addition, the cells can inhibit the activation of auxiliary T cells with NF-kβ inhibit the transcription of co stimulatory molecules, stop CD40L molecules on auxiliary T cell membranes, inhibit reactions of auxiliary T cells and MHC-I molecules antigen. More and more evidence show that CD8+CD28-regulatory T cells have relationship with allergic inflamation in asthma and maintain the peripheral organism immune tolerance. Upsizing CD8+CD28-regulatory T cells quantity or function in patients with asthma and correcting its immune disorders will become the new strategy for asthma.Myeloid derived suppressor cells (MDSC) is a kind of myeloid cells being closely linked with tumor immunity escape in cancer patients, compose of the different differentiation phase myeloid cells such as mononuclear cells、basophile, immature cells etc. As early as in1970, Italian oncologists failed to use cancer vaccines for tumor curing. After more than10years, they continue the study in the United States, found MDSC with suppression effect of immune in tumor-bearing animals. At the end of the90s, Gabrilovich studied the mechanism, they found that MDSCs exist in the spleen of the tumor animals, this group of cells exceeds40%,3-5times as much as that of normal person and the number of dendritic cells is decreased obviously. Huang found that MDSC induced the expression of regulatory T cells Foxp3in tumor-burdened mice, raising the proportion of CD4+, CD8regulatory T cells can cause immune incompetent and inhibition.In the experiment, the tumor-bearing mice as the object of study, using two magnetic bead separation to purify, activate and culture MDSC in vitro th rough adoptive transfer to intervent asthma animal airway inflamation directly, testing mice peripheral blood CD8+CD28-proportion. To confirm whether MDS C can interfere with asthma attack, we observed the regulation of CD8+regula tory T cell by MDSC, to explore the possible mechanism of action.Part one Establishment of the asthmatic mouse modelObjective To establish a BALB/c mouse asthmatic model by OVA sensitization and rechallenge.MethodsSPF female BALB/c mice (4to6weeks),18-20g, were purchased from Laboratory Animal Services Center of Southern Medical University, and maintained on an ovalbumin-free diet. The mice were randomly divided into2groups:control group and asthmatic group. Mice were sensitized on days0、14and21by an intraperitoneal injection of a mixture containing2mg of ovalbumin and5mg of Al (OH)3in saline (a total volume of0.2ml). At28days after the first immunization, the animals were firstly challenged with intranasal drop of1%ovalbumin, then challenged by exposure to an aerosol of2%ovalbumin(a total volume of40ml) generated by an ultrasonic nebulizer for30min. The30-min rechallenge procedure was repeated once each day from day28to day35. The control animals were treated with5mg of Al(OH)3in saline and rechallenged with saline solution. On the seventh day animals were then sacrificed. Six mice from each group were subjected to bronchoalveolar lavage. The bronchoalveolar lavages were stained with HE and total cell number and the number of eosinophils and neutrophils in bronchoalveolar lavage fluid were counted.Lung Tissues from other mice were sliced and HE stained for histological examination, including morphology, epithelial injury, perivascular and peritracheal inflammation infiltration of eosinophils after conventional drawing materials, dehydration, paraffin embedding, preparation paraffin section, HE dyeing, neutral gum sealing piece.Results1. The behavior changes after rechallenge Mice in asthmatic group displayed various types of allergic responses, such as scratching head and face, dyspnea, oral lip cyanosis, muscle spasm as well as gatism, while control group showed no such symptoms.2. The changes in total cells and different cell types in BALF②The total BALF cell number in the asthmatic group was (6.75±2.70)×104/ml, that is significantly higher than that in the control group(34.05±8.02)×104/ml (t=-9.557, P=0.006)②The number of BALF eosinophils in the asthmatic group was(10.85±3.14)×104/ml, that is significantly higher than that in the control group(1.35±0.96)×104/ml (t=-8.378, P=0.004)③The number of BALF neutrophils in the asthmatic group was(9.78±2.65)×104/ml, that is significantly higher than that in the control group(1.56±0.82)×104/ml (t=-9.450. P=0.002).3. Histological analysisMassive leukocyte was present in the lungs from the asthmatic mice. Neutrophils, eosinophils and lymphocytes were mainly found in the alveolar, interstitial and peribronchial regions. Goblet cell hypertrophy, epithelial damage, mucus hypersecretion, mucus plugs, as well as collagen deposition were also easily identified. Lungs in the control mice displayed a clear airway structure without any cell or collagen deposition.ConclusionsBALB/c mice first sensitized by an intraperitoneal injection of a mixture containing ovalbumin and Al(OH)3, and then rechallenged with intranasal ovalbumin inhalation in combination with exposure to an aerosol of ovalbumin successfully developed asthmatic clinical characteristics and pathological changes, which is consistent with airway pathophysiological changes during asthma attack. Part two The effect of myeloid derived suppressor cells derived from tumor-bearing mice on the CD8+CD28-regulatory T cells and airway in mice asthma modelObjectiveTo investigate the effect of Myeloid derived suppressor cells on the CD8+CD28-regulatory T cells of peripheral blood and the airway in mice asthmatic model.MethodsThirty five SPF BALB/c mice were selected for this study.5male mice aged6weeks were used for preparing MDSCs. Thirty female mice aged six weeks were equally and randomly divided into normal control, model control and cell transplantation groups. The phenotypes and percentage of MDSCs were detected using flow cytometry. The tumor-derived MDSCs were separated from tumor tissue using a magnetic cell sorting system and cultured in RPMI Medium1640containing GM-CSF. The cell morphological characteristics were observed by light microscopes. The mice of group B and C were sensitized by OVA and then stimulated with nebulized OVA, Mice in the group C were intravenously administered MDSCs which purified by magnetic cell sorting system at10days after sensitization. The airway was evaluated by HE staining.The total cell number of bronchoalveolar lavage fluid, the number of eosinophils and neutrophils were counted. The fractions of CD8+CD28-T cell of peripheral blood were tested by flowcytometry to analyze the correlation between eosinophils(EOS)of BALF and CD8+CD28-T cell of blood in group C.Results1. The behavior changes after rechallenge Mice in group B displayed various types of allergic responses, such as scratching head and face, dyspnea, oral lip cyanosis, muscle spasm as well as gatism, while group A showed no such symptoms, group C had the symptoms occasionally.2、The changes in total cells, eosinophils and neutrophils in BALF①The total BALF cell number in group A was (6.75±2.70)×104/ml in group B was(34.05±8.02)×104/ml, in group C was (17.39±3.49)×104/ml, that is significantly higher than that in the group A (P<0.01).C group was significantly lower than the B group (P<0.01)②The number of BALF eosinophils and neutrophils were(9.78±2.65)×104/ml and (10.85±3.14)×104/ml1in group B,(4.16±1.53)×104/ml and (6.99±2.07)×104/ml in group C.that were also significantly higher than those in the group A (1.35±0.96)×104/ml and (1.56±0.82)×104/ml (P<0.01).C group was significantly lower than the B group (P<0.01).3. Histological analysisLungs in the group A displayed a clear airway structure without any significant cell. Massive leukocyte was present in the lungs from the group B mice. Neutrophils, eosinophils and lymphocytes were mainly found in the alveolar, interstitial and peribronchial regions. Goblet cell hypertrophy, mucus plugs. In Group C,compared with group B,inflamation of bronchial submucosal, blood vessels and the surrounding lung reduced significantly.4Flow cytometry detecte peripheral blood CD8+CD28-T cells:There is a significant difference between the three groups of statistical significance (F=9.186, P=0.002)。Flow cytometry detecte peripheral blood CD8+CD28-T cells proportion of mice, asthmatic group (6.01±1.88)%, reduced significantly compared with control group (11.06±3.85)%, cell transplantation group(8.85±2.09)%, Difference have statistical significance (F=11.791,P<0.05). There are no statistically significant between Cell transplants and control group (P=0.336). 5EOS of BALF in cell transplantation group compare with peripheral CD8+CD28-T cells for relevant analysis.Pearson correlation analysis shows:BALF of eosinophil count compared with peripheral CD8+CD28-T cells percentage is a significant negative correlation in cell translate group.(r=0.709, P=0.022).Conclusion:MDSCs via intravenous influsion can up-regulate CD8+CD28-regulatory T cells of peripheral blood in asthmatic mice and effectively prevent the development of airway inflamation. |
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