Therapeutic Effect Of Human Umbilical Derived Mesenchymal Stem Cell On Murine Model Of Bronchiolitis Obliterans Like

Part Ⅰ:Establishment of a murine model of bronchiolitis obliterans by administration of diacetyl:a comparison of oropharyngeal aspiration and intratracheal instillationObjective:To compare the effects of different administration methods of diacetyl(DA)in the development of a mouse model of bronchiolitis obliterans(BO).Methods:SPF grade C57BL/6 male mice(6 to 8 weeks)were randomly divided into four groups with 10 mice in each group:oropharyngeal aspiration group(OPR),intratracheal instillation group(ITI),Control group(OPR-CON and ITI-CON).OPR group treated with DA(400mg/kg,327mg/kg)by oropharyngeal aspiration;ITI group received DA(400mg/kg,327mg/ml)through intratracheal instillation;OPR-CON group and ITI-CON group treated with sterilized distilled water instead of DA,the rest of the experimental conditions were same with the OPR and ITI group.The mice were kept in SPF class animal center for 7d to collect specimens.Collected bronchoalveolar lavage fluid(BALF)and the left lung were examined for pathologic examination.Results:Administration of DA by ITI or OPR resulted in airway injury detachment,lumen occlusion,and infiltration of inflammatory cells into the airway and surrounding vessels.The mortality rate was high in both the OPR and the ITI groups.Airway hyper-responsiveness,lung fluid neutrophilia,collagen deposition were also detected in the OPR and ITI groups.Intestinal epithelial tissue shedding was observed only in the OPR group.Conclusions:Administration of DA by ITI was demonstrated to be effective in developing a murine model of BO in C57BL/6 mice.Part Ⅱ:Interventional Effects of Human Umbilical Cord Mesenchymal Stem Cells on Obstructive Bronchiolitis in mouse ModelObjective:To observe the therapeutic effects of HUC-MSCs on BO murine model and to elucidate the mechanisms of these effects briefly.Methods:SPF C57BL/6 mice were randomly divided into 6 groups(n= 10):the blank control group(Control + NaCl),security control group(Control + HUC-MSCs),model group(BO + NaCl),treatment group(BO + HUC-MSCs,BO+ HUC-MSCs-CM and BO + CM).After being treated with DA for 1 h,NaCl,HUC-MSCs,HUC-MSCs-CM and CM was administered at a total volume of 200ul by tail vein.HUC-MSCs were labeled with Dil and their colonization in the lung tissue was observed.The mice were dynamically observed at the same time each day for survival,respiratory status and body weight changes.Trachea and lung tissues were collected on 3d and 7d for HE staining and masson staining.The expression of E-cadherin and CK-5 in the trachea and lung was detected by Immunofluorescence and Western Blot.Immunofluorescence was used to detect the colonization of Dil-labeled HUC-MSCs in the lung and trachea.Results:After intervention of HUC-MSCs and HUC-MSCs-CM,the general condition of BO mice was improved,the weight reduction tendency was significantly slowed(P<0.05),and the mortality rate was significantly lower(P<0.05).Inflammatory infiltration of trachea and lung tissue was decreased.Airway epithelial cells and basal cells were significantly repaired at 3 and 7 days.Immunofluorescence and western blot of tracheal tissue showed that the expression of E-cadherin and CK-5 in airway epithelial cells in BO + HUC-MSCs and BO + HUC-MSCs-CM groups was significantly higher than that in other groups(P<0.05).HUC-MSCs were labeled with Dil,and then their distribution in mouse lung tissue was followed.It was found that HUC-MSCs disappeared within 2 days in the trachea and the lung tissue gradually decreased after 4 days.Conclusions:HUC-MSCs and its conditioned medium could repair airway epithelial cell.It may be related to the repair of epithelial damage through the paracrine pathway.