Effect Of Human Umbilical Cord Mesenchymal Stem Cells Intervention On Murine Model With Acute Bronchiolitis Obliterans

PART I THE ESTABLISHMENT OF MURINE MODEL WITH ACUTE BRONCHIOLITIS OBLITERANSObjective:To explore the establishment of murine model with acute bronchiolitis obliterans.Method:C57BL/6 mice (SPF) were randomly divided into control group and BO group with 6 mice in each group. Mice in BO group were instillated intratracheally with Diacetyl (DA) (490 mg/mL,500 mg/kg) and mice in control group were treated with sterile distilled water. Other experimental conditions and methods in each group were the same. All mice were raised in SPF grade room. Three and seven days after instillation Enhanced Pause were detected and bronchoalveolar lavage fluid were collected; Then mice were sacrificed to remove left lungs for pathological observation. Mice were dynamically observed for general condition, body weight.Results:The model group mice were given DA (500 mg/kg) after one day and they presented wheezing, rapid breathing and other symptoms, the weight was significantly lower than the control group (P<0.05). Compared with the control group, the lung function (aerosolized methacholine concentration was 50 mg/ml) Penh increased significantly (P<0.05). Model mice inflammatory cell count and classification counting, neutrophils were significantly higher than the control group (P<0.05) at three days, the total cells and lymphocytes, macrophages were significantly higher than that of the control group (P<0.05) at seven days. Pathological section HE staining showed typical BO changes, around the 3 day model group bronchial lumen and infiltration of inflammatory cells, luminal severe occlusion, tissue in the acute stage of severe bleeding; 7 days of basal cells were severe necrosis, airway epithelial cells were attached to the substrate, but seen obvious cell hyperplasia and hypertrophy, cell core lost seriously, inflammatory cell infiltration in the lumen, the lumen wall thickening, fibrosis.Conclusion:The mouse model of bronchiolitis obliterans(BO) could be established successfully by intratracheal instillation DA (500 mg/kg) once.PART II EFFECT AND MECHANISM OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTERVENTION ON MURINE MODEL WITH ACUTE BRONCHIOLITIS OBLITERANSObjective:To observe the effects of HUC-MSCs on BO mice model and to illustrate the molecular mechanism briefly.Method:C57BL/6 mice were divided into five groups (n=8), (Control+NaCl, as blank control group; Control+HUC-MSCs, as security control group; BO+NaCl, as model group; BO+HUC-MSCs, as treatment group; BO+HUC-MSCs-CM, as treatment group. Saline, HUC-MSCs, and HUC-MSCs-CM (total volume was 200ul) were injected into mice by tail vein after 1h, respectively. HUC-MSCs were labeled by Dil, then tracking its distribution in lung tissue. The mice were kept in SPF class animal center to 3 and 7 days to collect specimens. They were dynamically observed the general status, body weight, pulmonary function, as well as the pathological and H&E staining for lung slices. Using immunohistochemical fluorescence to detect E-cadherin, CK-5 expression in trachea and cell immunofluorescence to detect E-cadherin, CK-5 expression in HUC-MSCs; Using immunohistochemical fluorescence to seek the colonization of HUC-MSCs in lung and trachea.Results:After HUC-MSCs intervention, the mortality rate of BO mice reduced significantly (P<0.05). Pathology of AEC and basal cells in lung tissue were repaired significantly within BO+HUC-MSCs and HUC-MSCs-CM group on 3 and 7 days. Less alveolar and bronchial inflammatory cells, alveolar septum. acute bleeding reduced clearly(P<0.05). Using Immunofluorescence sought airway epithelial repair in stem cells and culture media treated group significantly (P<0.05). Stem cells themselves did not express E-cadherin, CK-5. HUC-MSCs were labeled by Dil, then tracking its distribution in lung tissue of mice. In trachea, HUC-MSCs disappeared within 2d, and gradually reduced in lung tissue after 4d.Conclusion:HUC-MSCs could repair AEC in acute phase of BO mice. It might be related with immune regulation; or inhibit the expression of inflammatory cytokines of the damaged area micro-environment after the intervention of HUC-MSCs.