Effects Of Human Amniotic Mesenchymal Stem Cells(hAMSCs) Transplantation On Airway Inflammation And Remodeling In A Mouse Model Of Asthma

Part One Preparation of a mouse model for asthmaObjectives:To prepare a asthmatic model in mice by hypodermic injection combined with intraperitioneal sensitization and challenging with ovalbumin(OVA).Methods:Fourteen female BALB/c mice were assigned randomly to asthma group(AS, n=7) and saline control group(NS, n=7). Two groups were respectively sensitized on days 1 and 13 by hypodermic injection combined with intraperitioneal of OVA and normal saline, challenged with 10% OVA or normal saline repeatedly for five days on the 19 th day. Mice were sacrificed 24 hours after the final challenge. The bronchoalveolar lavage fluid(BALF) were collected for the total number of cells and classification. IL-5, IL-12, IL-17 levels were detected by ELISA, lung tissue pathological changes were observed by HE staining, airwaygoblet cells and mucus substance changes were observed by AB-PAS, collagen deposition area was observed by VG staining, and lung tissue Muc5 ac protein and m RNA expression levels were detected by i m m u n o h i s t o c h e m i s t r y s t a i n i n g a n d r e a l- t i m e q u a n t i t a t i v e P C R r e s p e c t i v e l y.Results:The total cell count and differential cell counts, expression levels of IL-5, IL-17 in BALF, the score of airway inflammatory infiltration, airway wall thickness, the positive staining area of hyperplasia of goblet cells, mucus material secretion in the airway wall and the collagen deposition, the expression of Muc5 ac protein, and Muc5 ac m RNA in lung tissue in AS group were significantly higher than those in NS group(P<0.01). The expression level of IL-12 was lower than the NS group, and the difference was statistically significant(P<0.01).Conclusions: Hypodermic injection combined with intraperitioneal sensitization and challenging with OVA can successfully duplicate the mouse model of asthma.Part Two Isolation, cultivation and identification of human amnion mesenchymal stem cellsObjectives: To isolate, cultivate and identify the human amnion mesenchymal stem cells from human amniotic membrane.Methods: The cesarean full-term fetal placenta was collected, h AMSCs were digested and isolated by mechanical combined with trypsin-collagenase and subcultured. T h e m o r p h o l o g y o f P 1 ~ P 3 c e l l s w a s o b s e r v e d. T h e p h e n o t y p e c h a r a c t e r i s t i c s of h AMSCs were identified by flow cytometry.Results: h AMSCs showed the typical adherent growth, fibroblastic and long spindle shape. High-level expression of CD90, CD105, CD44 and CD73 was observed in the third generation of h AMSCs, and almost no expression of CD34, CD11 b, CD19, CD45 and HLA-DR was seen.Conclusions: h AMSCs are successfully isolated and cultured in vitro.Part Three The effects of human amniotic mesenchymal stem cells(h AMSCs) transplantation on airway inflammation and remodeling in a mouse model of asthmaObjectives: To observe the effects of h AMSCs vein transplantation on airway inflammation and remodeling in asthmatic mice.Methods: Thirty five female BALB/c mice were randomly separated into five groups as fol lowings(n=7): saline control group(NS), asthma group(AS), human amniotic mesenchym al stem cells treatment group(AS+h AMSCs), dexamethasone treatment group(AS+DEX) and human amniotic mesenchymal stem cells control group(NS+h AMSCs). All groups exc ept NS and NS+h AMSCs were sensitized and challenged with OVA as described in Part One. In group s AS+h AMSCs and NS+h AMSCs, the third generation of h AMSCs suspensi on(1×106/ml) 0.25 ml were injected through tail vein, which were labeled with Brdu-labeled after 18 days of the first sensitization. And the other treatments were the same as grou ps AS and NS. In AS+DEX group, each mouse received dexamethasone(2mg/kg) i.p. 30 min before challengin. Mice were sacrificed 24 hours after the final challenge. The total n umber of cells and classification of irrigation were counted, and the level of cytokines IL-5, IL-12, IL-17 in BALF were detected by ELISA, Brdu marked in vivo colonization rate and h AMSCs homing ability in lung tissue were observed by immunofluorescence stainin g. HE, AB-PAS,VG staining, IHC and RT-PCR were used as described in Part One.Results: h AMSCs were detected in lung tissues of two groups of mice, and cell colonization in group AS+h AMSCs was obvious. The total cells, percentage of Eosinophile cell, IL-5, IL-17 and the score of airway inflammatory infiltration, the relative positive staining area of airway goblet cells and mucus and the collagen deposition, Muc5 ac protein and Muc5 ac m RNA expression in group AS+h AMSCs were lower than those in group AS and higher than those in group NS, and the differences were statistically significant(P<0.01). The expression level of IL-12 was higher than that in group AS, but lower than that in group NS(P<0.01). However, there was no significant difference between the NS+h AMSCs group and the NS group(P>0.05).Conclusions: 1. Exogenous h AMSCs can be homing to damaged lung tissues, especially in AS+h AMSCs group was significantly. 2. h AMSCs intervention in asthmatic mice can relieve the airway inflammation and remodeling possibly by regulating the balance of Th1/Th2 c e l l s a n d i n h i b i t i n g t h e f u n c t i o n o f T h 1 7 c e l l s a n d r e p a i r i n g d a m a g e d c e l l s.