Adipose-derived Mesenchymal Stem Cell (ADSC) Regulate Immune States And Repair Mucosal Injury Of Allergic Rhinitis Mouse Model

Background and ObjectiveAdipose stem cell?ADSC?transplantation was performed on a mouse model of allergic rhinitis to observe changes in inflammatory response of the nasal mucosa and mucosal function repair.Methods1.Isolation and culture of ADSCTen SPF Balb/c mice were sacrificed and killed after cervical dislocation.Sterile adipose tissue was taken from the groin and epididymis,digested,resuspended,inoculated,and cultured at 37°C in a 5%CO2 incubator.After 24 hours,the medium was changed half-time for the first time,and the medium was replaced every 2³ days.After the cell fusion reached approximately 80%,it was digested with trypsin.2.Preparation of AR mouse modelTen-week-old SPF Balb/c mice,18²5 g,were sensitized on days 1,7,14 and21 via an intraperitoneal injection of ovalbumin?OVA?suspension[1 mg/mL OVA,20 mg/mL aluminum hydroxide?Al?OH?₃?mixed in normal saline],200?L/per mouse.After 3 weeks,all animals were challenged by the daily nasal instillation of OVA suspension?100?gOVA mixed in 20?L normal saline?using a micropipette to day 35.The symptoms of the mice were observed within 15 min after nasal instillation and recorded.3.Injection of ADSC via tail veinThe mice were divided into three treatment groups of 12 mice each,as follows:control group A?PBS/PBS/PBS?,stem cell intervention group B?OVA/OVA/ADSC?,allergic rhinitis group C?OVA/OVA/PBS?.Rat tail vein injections were performed on the 22nd to 25th days after challenge respectively.The symptoms of mice in each group were recorded within 15 minutes after the last challenge.The mice were sacrificed 24 hours later.Peripheral blood and nasal mucosa were collected.Histological changes were observed by HE and PAS staining,and the levels of serum IL-4 and IFN-?were detected by enzyme-linked immunosorbent assay?ELISA?.The nasal mucosal levels of mRNAs encoding INF-?,IL-4,IL-9,IL-17 and Foxp3 in nasal mucosa of mice in each group was detected by quantitative PCR.Immunohistochemistry was used to detect the level of?-tubulin IV,ZO-1,MUC5AC.Statistical analysis of the data.Results1.Isolation and culture of ADSC in mice24 hours after inoculation,a small amount of cells adhered to the dishes,the size and shape are not uniform,showing a short spindle type,polygonal shape,poor refraction.After 48 hours,the number of adherent cells increased and gradually extended,showing a long spindle.Identification of ADSCs by flow cytometric surface markers:CD29?+?,CD44?+?,CD34?-?.Immunofluorescence results:CD90?+?,CD34?-?.2.Effect of adipose stem cells on mice with allergic rhinitisCompared with the control group,mice with allergic rhinitis often scratched the nose,accompanied by continuous sneezing,and had clear watery nose.Nasal mucous membranes are largely shedding.There were edema of the interstitial tissue,expansion of small blood vessels,and changes in cell morphology.The number of eosinophils and goblet cells increased significantly.The levels of serum IL-4,IFN-?increased.The nasal mucosal levels of mRNAs encoding IL-4,IL-9,IL-17,IFN-?increased of nasal mucosal,while Foxp3 decreased.The level of?-tubulin IV,ZO-1decreased in immunohistochemistry,but the level of MUC5AC increased.Increased nasal rubs and sneezes were diminished by treatment with ADSC.Eosinophilic infiltration and the number of goblet cells decreased significantly in ADSC intervention group compared to mice with allergic rhinitis.The levels of serum IL-4,IFN-?were significantly decreased in ADSC intervention group than the AR mice.Compared with AR group,the nasal mucosal levels of mRNAs encoding IL-4,IL-9,IL-17 and IFN-?were decreased and Foxp3 were increased in the ADSC intervention group.And the immunofluorescence of?-tubulin IV and ZO-1 was slightly increased,while that of MUC5AC was decreased.The above differences were statistically significant?P<0.05?.ConclusionADSC can eliminate allergic symptoms,reduce the infiltration of eosinophilic and inflammatory cytokines and the number of goblet cells in nasal mucous membrane of AR mice,delay the remodeling of epithelium,and restore epithelial function.