| Asthma is caused by a variety of cells including airway inflammatory cells,airway structural cells and cell components. It’s a chronic inflammatory respiratorydisease.Patients often wheeze recurrently caused by inhaling allergens. That can doharm to lung function.And that also brings heavy psychological and financial burdento the children and their families.Asthma symbolized with airway inflammatory cellsinfiltration,goblet cells metaplasia, increased mucus secretion, airway lumen blocked,airway wall thicken, collagen fibers proliferate,type2helper cells excessiveactivation, ariway hyper-responsiveness (AHR), airway remodeling and resrumimmumoglobulin (IgE)elevated.Currently it has always been believed: sentiveindividuals abnormally immune response to external environmental antigen can causeasthma.Immune disorders play a central core role in the pathogenesis of asthma.During the past more than20years,Studies suggested that Th1/Th2celldifferentiation imbalance,Th2cell-dominated immune disorder is an important factorin the formation and development of allergic asthma.Recently discovered thatTh1/Th2cell differentiation imbalance can not explain the whole asthma pathogesis,itis only the superficies of asthma.Asthma underlying reason is the body’s immunetolerance to the allergen was broken, However the Th2/regulatory T cells imbalanceplay an important role in the broken of body’s immune tolerance to the allergen. Inaddition,Th17cells is a new type cells that can influence the immune response of asthma,which is discoverd recently.Researches on animal models of asthma foundthat changes in expression levels of Th17cells or Treg cells could afflect asthmaairway inflammation and airway reactivity.Its pathogenesis is so complex and still notfully understood. Though it has been discovered that many new type cells,cellcomponents,involed in the pathogenesis of asthma,and proviede a new theoreticalbasis to asthma diagnosis, treatment, prevention.Currently asthma still can not becured completely.There still existed some ineffective refractory asthma which can notbe cured by comprehensive treatment,including inhaled corticosteroids.Thatprompted us to make further research for the pathogenesis of asthma.Myeloid derived suppressor cells (MDSCs) are a heterogeneous class of bonemarrow cells mainly including immature macrophages,immature dendritic cells andimmature granulocytes.They lack the mmature characteristic markers that expressedon the mature macrophages, mononuclear cells and dendritic cell surface.Thecommon symbol for the surface of human surface cells is Lin-HLA-CD33+orCD11b+CD14-CD33+.And on the cell surface of mice is CD11b+Gr-1+, and in micewe can identify accurately MDSCs by using the Gr-1antibodies against-antigensreactions. MDSCs could play an important role in the regulation of adaptiveimmunity, inflammation and innate immune system in body. That could cause tumorimmune escape, immune-related diseases and chronic inflammatory diseases.Overseas studies have shown that MDSCs are highly expressed in the lung allergicinflammation and is involved in asthma lung inflammation,airway hyper-responsiveness and airway remodeling.Through the past research we found thatMDSCs of asthmatic children in peripheral blood was significantly higher than thecontrol group of children, show MDSCs may be involved in the pathogenesis ofasthma. And the study found that their immune function can be related to theirsynthesized and secreted high levels of arginine synthase and inducible nitric oxidesynthase.Inducible nitric oxide synthase is not expressed under physiological conditions,when the body is infected, can induce the expression of iNOS. And then sythesize alot of NO. High concentrations of NO can produce a series of proinflammatory.It hasbeen reported that endogenous NO is usually found in animal and human exhaled,exhaled NO(eNO) concentration increases in asthma,further increased whenasthma worse. Also plasma NO in acute asthmatic children was significantly higherthan those in asthma remission chiledren and normal children, suggesting thatendgenous NO may be invoved in the pathogenesis of asthma.And now we also knowthat excessive NO in the lung tissue is mainly synthesised by inducible nitric oxidesynthase.Bronchial asthma pathogenesis is so complex, and it is not entirely clear now.MDSCs is a group of newly discovered populations of cells that can regulate immunefunction.In present study suggests that is more concentrated in the tumor, severeinfections, sepsis fields. Asthma and cancer, as there is a serious immune disorderpathogenesis, we might imagine there is a certain link between MDSCs and asthma.iNOS excessive expression then generate an excess of NO can be one of theimportant mechanisms causing inflammation. iNOS/NO system can play animportant role in the pathogenesis of asthma.Appropriate intervention of iNOSexpression can reduce inflammation,also can change the asthmatic response. GivenMDSCs can synthesize and secrete high arginine synthase and iNOS play animportant role in cancer, sepsis, autoimmune diseases. But asthma also immunedisorders, whether MDSCs highly synthesize and secrete arginine and iNOS exist inasthma, this mechanism is not known. In this study, we established mice model ofasthma,and detect the expression levels of MDSCs and iNOS in asthmatic mice,andattempts to explore the relationship between the two with asthma, and broadened thestudy the pathogenesis of asthma.We try to provide a new theoretical basis for thediagnosis and treatment of asthma.ObjectiveWe established asthma mice model, and detect expression levels of myeliod-derived suppressor cells (MDSCs), inducible nitric oxide synthase in the lung tissue,Further investigate their roles of both in asthma, aiming at broadening the horizons of asthma pathogenesis.Materials and Methods1Experimental mice groups and asthma model makingExperimental mice were purchased from the Experimental Animal Center ofHenan Pronince.30clean-level,healthy,female,6weeks,BALB/c mice (Experimentalanimal license number: SCXK (Henan)2010-0002),adaptive feeding for one week inthe Third Afflliated Hospital of Zhengzhou University Research Center,they eat anddrink freely during that time.Before the experiment，we measure the mice body massis20-25g by electronic scales.The mice were randomly divided into three groups: theAsthma model Group the budesonnide Group and normal saline control Group. Eachgroup has10mice. Model sensitization phase：The asthma group and budesonnideintervention group,on the day of1,8,15of the experiment,each mice had beenintraperitoneal injected of antigen mixture(0.02mg OVA aluminum hydroxidepowder2mg plus saline to0.2ml). From the22th day,1%OVA is inhalated toexcite,30minutes each excitation,once daily,14times totally. Intervention Groupsenitized and excited in the same way with Asthma Group. The difference betweenthe two groups is that before each excition, inhalated1mg(2ml) Budesonide,30minutes for the Intervention Group.Control Group was sensitized and excited withsaline,its methods and dose was the same with Asthma Group.Mortality were notoccured in the three groups of mice during the experiment.2collection of mice lung samplesWe anesthetize all the mice by Inhaling Ether in each group at the end of the last24h inhalation, and then fixed the mice on the operating table,Open the chest, Fullyexpose the mice heart and lung, ligature the left lung hilum,take the left lung tissue,place into-80℃refrigerator,keep as RT-PCR specimens,used to detect theiNOSmRNA expression level.Immediately followed we Intubate to the pulmonaryartery by the right ventricle. We syringe the right lung lobue repeatedlyand quicklywith saline.when it turned white, we syringe the4%formaldehyde solution to the lung tissue, aimed at internal fixation.Take it, then external fixate the lung tissue morethan48hours by the4%formaldehyde solution. Then embedded in paraffin,cutconsecutively into3μm thickness slice, HE staining, immunohistochemical stainingfor next analysis.3HE stainingTake the4%neutral formalin fixed right lung tissue, Embedded, sliced, grilled,dewaxed,dehydrated, then HE staining. Observe airway structural changes andinflammatory cell infiltration, and measuring the same level of bronchial wallthickness.4immunohistochemisry stainingWe use the SP method for immunohistochemisty staining.Dewaxed, dehydratedand antigen repaired, endogenous peroxide sealed. Add anti-Gr-1(1:200) antibody,incubate at4℃over night.Next day, add the sendory antibody,comply withimmunohistochemical staining steps.Observed the results under a microscope at highmagnification (10×40) vision,pictured. Each specimen taken five randomly selectedhigh-power microscope field of view, then applicate BI-2000medical pathologicalimage analysis system analyzes the results.Take the mean IOD of five horizonspositive reaction as a measurement value of the group.5RT-PCRTake the lung tissue (100mg) from Refrigerator of-80℃, extracting the totalRNA from lung tissue by Trizol method. Add the reverse transcription reagents,reverse-transcribed into cDNA. Add the designed iNOSmRNA primers, amplificatethe objective gene.The product was subjected to Gel electrophoresis on an agarose gelafter the amplification.Using gel electrophoresis systems to determinate iNOSmRNAand GAPDHmRNA bands gray value, respectively.6Statistics analysisUtilize SPSS17.0for statistical analysis. We use (x±s) to express the measurement data.The differences among the three groups was analized by ANOVA，Pairwise comparison using LSD-t test,α=0.05. Correlation analysis between twoquantitative variables was analyzed by Pearson linear correlation,α=0.05.Results1Pathological changes in mice lung of each groupWe observe the changes at high magnification（10×40）field by bio-opticalmicroscope. Asthma group：airway wall thickening，luminal mucus and epithelialcells filled in the airway,stenosis,airway wall goblet cell metapalsia,mucus excessivesecretion,collagen fibers proliferating,inflammatory cells infiltration. InterventionGroup:it aslo exists above changes, compared with asthma group,but some relief.The Control Group: nomal airway wall thickness,no lumial stenosis,no goblet cellmetaplasia,scattered inflammatory cells.As Figure A,B, C.2Immunohistochemical resultsWe found that Gr-1positive MDSCs appear brown after immunohistochemicalstaining, expressed in alveolar interstitium and around the bronchial and blood vesselwall. Asthma Group average (155.35±15.51).Intervention Group’s expression wasweaker than Asthma Group,its average (129.32±8.91). Control group average(112.27±4.65). we saw a statistically significant difference among them,whencompared.3RT-PCR resultsAsthma Group band is the brightest，and its gray average was0.637±0.051，Intervention Group’s brightness was weaker than Asthma Group,its gray average0.347±0.033. The control group was only0.178±0.022,that has the weakest gray.4Coralation Analysis between MDSCs and iNOSThe MDSCs Results and the iNOSmRNA results have a positive coralation inthe asthma group. The correlation coeffietient is0.667, P value is0.031. Conclusions1.MDSCs, iNOS may be involved in asthma airway inflammation.2.MDSCs may promote the expression of iNOS gene, then play a role in airwayinflammation.3.Budesonide may inhibit the expression of MDSCs, iNOSmRNA, then improveinflammation. |
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