| Objective:Estblishing BALB/c mice models for atopic dermatitis(AD)to detect the expression of EDC-related proteins S100A7,LOR,IVL of skin lesions.It is helpfμL to explore the expression of EDC-related proteins in AD skin barrier dysfunction.To further clarify the feasibility of the AD animal model and the relationship between epidermal barrier dysfunction and the pathogenesis of ADMethods:In this study,40 BLAB/c female mice of SPF grade with 6-8 weeks old and about 16-20g were selected and randomly divided into such 2 groups as AD group(AD group,n=20)and normal control group(C group,n=20).The 2 groups mice were removed back hair(squre:8cm2)by 8%sodium sulfide amylum solution from a week before the experiment.The methods of sensitizing and stimμLating AD group:smearing 0.5%2,4-Dinitrofluorobenzene(DNBF)in acetone/olive oil(3:1)on mice back area with hair removal.On the first and second day of experiments,0.5%DNFB in acetone/olive oil(dose:100μL)were applied to mice back area with hair removal in AD group once a day.From the third to sixth day,there is nothing to do on the removal areas of these mice models.On the 7th day of the experiment,70 μLO.5%DNFB solution(solvent:acetone:olive oil 3:1)was applied to the same back skin area and thereafter twice a week for 10 times.Model group:The dose and time of each application were the same as those of the AD group.The acetone/olive oil solution was applied in the depilation area,the ratio was 3:1,but the solution did not contain DNFB.On the 35th day,the model was established.We observed the number of clapping in the two groups within 10 minutes and evaluated the severity of the lesion.Mice were anesthetized with ether,and their right eye was taken to remove blood.The supernatant was centrifuged and frozen in a refrigerator at-80 ℃.The body weight and the weight of the spleen were recorded in both groups.Take the skin of the back skin area for the production of pathological specimens and the purpose of protein detection.The levels of total IgE and IL-4 in serum were detected by ELISA.The expression of EDC-related protein S100A7,LOR and IVL in skin of two groups was detected by Western blot.Histopathological observation was performed by HE staining.ResμLts:1.Mice skin inflammation score:AD group skin inflammation score(9.00)higher than the normal control group(0.00),the difference was statistically significant(P<0.05).2.Spleen index and scratch count resμLts:The spleen index(2.12±0.27)in the AD group was higher than that in the normal control group(1.77±0.16),The number of scratching in the AD group(13.55 ± 1.71)was higher than that in the normal control group(1.13 ± 0.86),the difference was statistically significant(P<0.05).3.Histopathological test resμLts:Normal control group mice did not damage the back of the skin,microscopic observation of no abnormal structural changes,no inflammatory cell infiltration.The AD group of microscopic observation of the epidermis,dermis layer of varying degrees of thickening,part of the epidermis shedding,necrosis,showing a large number of inflammatory cell infiltration.4.Western Blot test resp.Lts:AD group S100A7/β-actin ratio(1.36±0.12)Was higher than the control group(0.77 ± 0.05),AD group LOR/P-actin ratio(1.22 ±0.19)higher than the control group(0.73 ± 0.09),AD group IVL/β-actin ratio(0.64 ±0.05)lower than the control group(1.07 ± 0.16),The difference was statistically significant(P<0.05).The correlation coefficients of S100A7/β-actin,LOR/β-actin,IVL/β-actin and skin inflammation were 0.579,0.504,-0.546,The difference was statistically significant(P<0.01).The correlation coefficients of S100A7/β-actin with LOR/β-actin and IVL/β-actin were 0.679 and 0.782,Differences were statistically significant.S100A7/β-actin and IL-4 correlation coefficient of 0.792,moderate positive correlation,the difference was statistically significant.5.ELISA test resμLts:The total serum IgE concentration in the AD group was(17.60 ± 1.77)μg/mL higher than that in the control group(12.39 ± 1.43)μg/mL,The serum total IL-4 concentration(86.64 ± 3.58)pg/mL in AD group was higher than that in the control group(57.11 ± 2.35)pg/mL,the difference was statistically significant(P<0.05).The correlation coefficients of total IgE and IL-4 in serum were 0.68 and 0.69,and the differences were statistically significant(P<0.01).Conclusion:1.The use of DNFB to establish an AD animal model is feasible and provides more possibilities for clinical tirials of AD.2.EDC-related proteins S100A7,IVL,LOR and the degree of inflammation of the skin,and there is a relationship between EDC internal proteins.3,Epidermal abnormalities caused by epidermal barrier dysfunction can affect the pathogenesis of AD. |
