| Background and Objective:Bronchopulmonary dysplasia?BPD?is a chronic lung disease,which lack of effective therapies to improve the outcome of this disease.We aim to confirm the therapeutic effect of Human umbilical cord-derived mesenchymal stem cells?UC-MSCs?on oxygen-induced model of BPD and to explore the effect of UC-MSCs on aberrant elastin remodeling,and to illustrate the molecular mechanisms of UC-MSCs effects on BPD neonatal rats.Methods:The first stage:Newborn rat pups were randomly divided into four groups:?1?Room air control??RA+PBS?:normoxia?21%O2?plus PBS was administrated by intra-tracheal route on postnatal 7?P7?.?2?Room air control II?RA+UC-MSCs?:normoxia plus UC-MSCs was administrated by intra-tracheal route on P7.?3?BPD model?O2+PBS?:hyperoxia plus PBS was administrated by intra-tracheal route on P7.and?4?UC-MSCs treatment?O2+UC-MSCs?:hyperoxia plus UC-MSCs was administrated by intra-tracheal route on P7.N=24.Then their lungs were all harvested at P21.For explore the therapeutic effect of UC-MSCs in studies,the survival curves were generated,and differences were evaluated.Body weights were calculated by the average body weight of a litter.Lungs of rats were used for hematoxylin and eosin?H&E?staining to observe the morphological structure of lung tissues,and bronchoalveolar lavage fluid?BALF?were harvested for the detection of total cell number count,classified count,protein concentration.The lung sections were stained Gomori's Aldehyde-Fuchsin method for elastic fibers and Immunohistochemistry for elastin protein,respectively.Quantitative real-time PCR and Western blot were applied to measure elastin expression,at the mRNA and protein levels,respectively.Using transmission electron microscopy,we further investigated the ultrastructure of elastic matrices under different conditions.Quantitative real-time PCR was also applied to measure the expression of gene related to elastin synthesis and assembly components and proteoglycans metabolism.We next used a spectrophotometric assay to determine the free neutrophil elastase?NE?activity in the lung tissues.And we also measure the expression of TGF-?1 protein and IL-1?gene.The second stage:To confirm the paracrine activity of UC-MSCs in BPD,UC-MSCs were transfected with lentivirus carrying the green fluorescent protein?GFP?gene,then harvested and prepared for intra-tracheal injection into the lung.Lungs were harvested at 1h,12h,24h,48h,and 72h after UC-MSC transplantation.Frozen lung specimens were sliced,and GFP fluorescence was analyzed by fluorescence microscopy.Then we applied the concentrated conditioned medium?CM?from UC-MSCs to treat rats with BPD.To explore the effects of UC-MSCs on elastin expression in vitro,we cultured HLF-a in presence of 90%O2 for 24 h,followed by coculture of O2-stimulated HLF-a with UC-MSCs for 48h to assess their relationship in vitro.Subsequently,cell lysateswere obtained for Western blot detection of the elastin protein and a-SMA,a myofibroblast marker.UC-MSCs were co–cultured with O2-stimulated HLF-a and supernatant was collected for detection EGF secreted by UC-MSCs.In vitro experiments,we used neutralizing antibody?NtAb?to block the function EGF,and after that CM of UC-MSCs were added into the O2-stimulated HLF-a to measure the expression of elastin protein and a-SMA.Data are mean±S.Differences between two groups were determined by Student's unpaired t-test.Multiple comparisons of parametric data?mRNA and protein levels,quantitative histological measurements?were performed by one-way ANOVA at specific time-points,followed by Bonferroni multiple test between group pairs.Survival curves were compared by the log-rank test.P<.05 was considered statistically significant.Results:The first stage:The survival rate was decreased by O2exposure,whereas UC-MSCs administration significantly increased the survival of the rats exposed to O2?P<.05?.Decreased weight gain was observed in the O2+PBS group on P14 and P21.UC-MSCs administration improved the weight loss in the pups exposed to O2?P<.05?.Small and united alveoli were found in the room air?RA?control group,alveoli in the O2+PBS group showed focal airspace enhancement and anomalous alveolar sizes,suggesting impaired alveolarization.These hyperoxia-induced morphological changes and the impaired alveolarization were ameliorated by UC-MSC transplantation.In morphometric analyses,the mean linear intercept?MLI?and septal counts were significantly altered in the O2+PBS group?53.32±2.728 mm in MLI;29.80±2.913 in septal count,P<.01.vs.RA group?.After UC-MSCs transplantation,hyperoxia-induced morphometric abnormalities were attenuated?43.86±2.035 mm in MLI;51.80±3.062 in septal count,P<.01 vs.O2+PBS?.Meanwhile,the lung injury was attenuated,the total cell count and neutrophil count and protein in BALF were deceased?P<.01?.Neonatal rats of the RA control group showed elastin deposition patterns,with accumulation to the tips of alveolar septa?with considerably less dispersion of elastic fibers?.80%of O2-injuried lungs revealed different elastin distribution patterns,with disorganized elastin fiber networks prominent throughout the walls of distal respiratory units and shortened,blunted secondary crests.Interestingly,after UC-MSC treatment,elastin distribution returned to normal.Our results revealed that elastin amounts in the lung parenchyma,both at the mRNA and protein levels,were increased in lungs of O2+PBS group compared with RA controls?p<0.01?,but decreased after UC-MSC treatment.We also tested expression of genes related to elastin synthesis and assembly,such as Fbn1,Fbn2,and Fibulin5.The results confirmed the functional importance of UC-MSCs in elastin metabolism and provided a plausible clue that UC-MSCs may be of value in the treatment of BPD by affecting elastin synthesis and assembly.And also suggest that impact on proteoglycans metabolism may account,at least in part,for beneficial effects of UC-MSCs administration in stabilizing elastin in the lungs of BPD.UC-MSCs effect the ultrastructure of elastic matrices,decrease the expression of TGF-?1 and IL-1?after hyperoxia,suppress TGF-?1 activation and inhibit the NE activity active?P<.01,vs.O2+PBS?.The second stage:UC-MSCs were concentrated in the trachea 1h post-injection,progressively infiltrated the lung parenchyma though the tracheal wall at 12h post-injection,and populated the whole lung parenchyma at 24h post-injection.However,UC-MSCs remained in the lung parenchyma at 48h post-injection,with much lower fluorescence intensity compared with that obtained at 24h post-injection.Additionally,GFP-labeled UC-MSCs almost disappeared at 72h post-injection,suggesting that UC-MSCs secrete soluble factors responsible for relieving hyperoxic lung injury in rats,rather than differentiating into target cells and the CM of UC-MSCs could improve animal survival and body weight?P<.01,vs.O2+PBS?;meanwhile,scanning electron microscopy of lung tissues demonstrated that the CM of UC-MSCs could ameliorate O2-induced lung injury in rats.Elastin mRNA and protein expression in O2-stimulated HLF-a were significantly reduced by UC-MSCs co-culture?P<.01?.In addition,a-SMA protein level in O2-stimulated HLF-a was significantly reduced by UC-MSCs co-culture?P<.01?,indicating that UC-MSCs could inhibit HLF-a transdifferentiation into myofibroblasts in vitro.In vitro cell results,when UC-MSCs were co-cultured with O2-stimulated HLF-a for 48h,the expression of EGF secreted by UC-MSCs was significantly increased.And blocking EGF in MSC-CM with Nt Ab can decreased the expression of EGF secreted by UC-MSCs.However,Blocking EGF in MSC-CM with NtAb,the therapeutic effect of MSC-CM was significantly decreased.Conclusion:Airway delivery of UC-MSCs rescues the oxygen-induced model of BPD,and the present findings suggest that UC-MSCs could ameliorate aberrant elastin expression in the lung of hyperoxia-induced BPD model.Meanwhile,UC-MSCs protect the O2-induced lung injury of BPD model primarily through paracrine secretion with EGF,which provides a novel evidence for clinical treatment of BPD by using UC-MSCs. |
PDF Full Text Request