Effects Of Differrent Sensitizing Doses Of Antigen On Immune Reaction Type,Airway Function And Pathogenicity In Murine Model Of Asthma

Objective: To study the effects of different doses of ovalbumin(OVA)for the type of immune response,airway function and Pathogenicity in murine model asthma.Methods : 42 SPF grade female Balb / c mice were divided into 7 groups of6 according to a random number table: group A(group 25μg),group B(group40μg),group C(group 80μg),group D(group 100μg),group E(group 200μg),group F(group 1000μg)and group N(control group);group A,B,C,D,E,F were asthma model groups.Sensitized the mice to product asthma model with different doses of ovalbumin(OVA): asthma model groups sensitized with different doses(20μg,40μg,80μg,100μg,200μg,1000μg)of Ovalbumin(OVA),and were injected intraperitoneally with 0.2ml suspension mixture of OVA and 4mg aluminium hydroxides gel for sensitization on the 0st day,7th dayand 14 th day;control group was sensitized was injected with 0.2ml suspension mixture of 4mg aluminium hydroxides gel on the Ost day,the 7th day and 14 th day.Begining from the 21 th day,Each asthma model groups inhaled 50 ml 1%atomizing OVA PBS suspension 30 min every other day for five times in a row.Observed mice’s behaviors and activity during atomization process.Detected airway responsiveness and lung function at the last time of Atomization,and compared the percentage of standard airway resistance(sRAW%)in each group.Count the total number cells and the percentage of eosinophils in bronchoalveolar lavage fluid(BALF).Detected the OVA-specific antibodies in serum IgE(OVA-IgE)levels with enzyme-linked immunosorbent assay(ELISA).Fluorescence quantitative polymerase chain reaction(PCR)to detect expression of interferon-y(IFN-y),interleukin-4(IL-4)and interleukin-9(IL-9)mRNA in lung tissue and compared the expression level in each group with ΔΔCT method.Observed the airway structure and pathological changes of mice lung tissue that treated with hematoxylin-eosin(HE)staining and periodic acid-Schiff(PAS)staining under the light microscope;and perform pathological semi-quantitative score to estimate the severity of airway inflammation and mucus secretion.Used SPSS 17.0 statistical software for statistical analysis.Results : In the process of sensitization and atomization,the mice showed typical symptoms of asthma;and group A,B and C showed more severe symptoms than the high-dose sensitized group of group D,E,F and N.The sRAW% of group A,B,and C higher than group D,E,F and N(P <0.05),and inlow-dose sensitized groups,the sRAW% of group B higher than group A and C(P <0.05).The total cells number in BALF of group A,B,C and D was significantly greater than group E and N(P<0.01),and the total number of group Bcells is greatr than group A,C and D(P<0.01),Group F cell count to zero;the percentage of Eos of group A and B was significantly greater than group C,D,E and N(P <0.01),and group B is higher than group A(P<0.01).Lung pathology results: group A,B,C and D shows a Symptom of asthma model-thickening of bronchial walls,increase of mucosal folds,hyperplasia of goblet,hypertrophy of smooth muscle,Mucus secretion and a lot of inflammatory cells infiltration around the airway;and group B show the most obvious symptom;the bronchial wall damage severely in group E and F,which could not form asthma model,airway epithelium was incomplete,epithelial cells shed seriously,muscle ruptured,the shape of airway deformitied,surrounded by a small amount of inflammatory cell and mucus secretion reduced in the lumen;with increasing dose of antigen,the airway damage more and more severely.Semi-quantitativeanalysis of pathology: airway inflammatory cell infiltration and mucus secretion in group A,B,C and D were more serious compared with group E,F and N(P<0.05),in these group,group B was the most serious(P<0.05).Level of OVA-IgE in group B serum was significantly higher than group A,C,D,E,F and N(P<0.01).Expression of IL-4 mRNA in low-dose group(group A,B,and C)lung tissue was higher than group D,E,F and N(P<0.05),and group B is greater than group A and C(P<0.05);expression of IFN-y mRNA of lung tissue6in group D and E was significantly higher than group A,B,C and N(P <0.05),and group E is higher than group D(P<0.01);expression of IL-9 mRNA ingroup B lung tissue is higher than group A,C,D,E,F(P<0.01)and group N(P<0.05).Conclusion: Low doses of antigen can form model of asthma,and high doses of antigen can not form model of asthma.40μg is the best sensitizing dose to form a murine model of asthma.Different doses of antigen cause different immune response type,different effect on organ function and different pathogenicity.low-dose antigen cause Th2 immune response resulting in aasthma model,while high doses cause Thl immune response resulting in suppression of asthma.