| ObjectivesTo investigate whether Edu-labeled autologous bone marrow mesenchymal stem cells can be expanded and differentiated into airway epithelial cells,vascular endothelial cells,etc.in lung tissue,and whether it can repair damaged lung tissue,and reconstruct lung function.Methods1.Healthy male New Zealand white rabbits were 2.7-3.5kg and 32 were divided into 27model groups and 5 healthy controls.The emphysema model was constructed by the method of nebulization with trypsin(2.5g/100ml Nacl).The emphysema model was sprayed once a week.After continuous atomization for 4 weeks,the feeding was continued for 12 weeks.After the completion of the model,the average lung volume was calculated by dual-source CT.The changes of lung volume before and after the modeling were compared between 8 weeks and 16 weeks after operation.The gross specimens of lung tissue and HE pathological changes between the contrast model group and healthy group were used to verify the pulmonary emphysema model.2.Randomly selected 18 rabbits were randomly divided into 6 groups,3 in each group.They were model control group,anti-oxidation group,low-dose stem cell treatment group,low-dose stem cell combined with anti-oxidation treatment group,high-dose stem cell treatment group,high-dose stem cell combined with anti-oxidation treatment group.0.5 ml of bone marrow fluid(each rabbit's bone marrow fluid was serially numbered for culture,amplification,Edu staining,and then self-introduced)was taken from the tibia of 12 model rabbits selected for stem cell therapy.Bone marrow mesenchymal stem cells were cultured by bone marrow adherence method.When the bone marrow mesenchymal stem cells were cultured to the third generation,bone marrow mesenchymal stem cells were co-incubated with 10 uM Edu for 48 hours.100 ul of Edu-labeled bone marrow mesenchymal stem cells were collected for fluorescent staining with Apollo488 and Hoechst 33342.The proliferation status of bone marrow mesenchymal stem cells was observed by fluorescence microscopy.Edu-labeled bone marrow mesenchymal stem cells(low-dose stem cells:4x10~6,high-dose stem cells:8x10~6)were transfused into rabbits through ear vein autograft.The antioxidant treatment group was simultaneously treated with antioxidation(dexamethasone0.3mg/kg intramuscular injection and reduced glutathione 40mg/kg intravenous injection for 7 consecutive days).The changes of average lung volume and electrocardiographic monitoring indexes(heart rate,respiratory frequency,oxygen saturation),plasma oxide H2O2 and inflammatory factors(TNF-,TGF-beta,IL-10,IL-1)were observed at 4 and 8weeks after bone marrow mesenchymal stem cells infusion.At the end of the experiment,oxidant and inflammatory factors were detected in the bronchoalveolar lavage fluid of each group.Lung tissue was stained with fluorescence staining to observe whether Edu positive cells and lung tissues were stained by HE.Results1.Experimental rabbit emphysema model identification:The average lung volume(68.3±10.7)cm~3 at 8 weeks after modeling,16 weeks(74.0±13.9)cm~3 after modeling and the mean lung volume(45.9±6.5)cm~3 before modeling.In comparison(P<0.05),there was a statistically significant difference in the mean lung volume between the two groups before and after modeling.The average lung volume gradually increased with time.This indicated that after the modelling of porcine trypsin finished,with the observation time prolonged,lung tissue continued to damage.The observation of gross lung specimens was that lung tissue became gray white after modeling,and also can see bulla bulge.The observation of HE staining was that healthy group alveolar structure was complete.The alveolar structure was intact in the healthy group,consisting of monolayer epithelial cells,without inflammatory cell infiltration.In the model control group,the bronchial epithelial cells fall off,the alveolar septum breaks,the alveoli fuse to form the pulmonary bullae,the pulmonary interstitial hyperplasia,and a large number of inflammatory cells infiltrate around the bronchioles,pulmonary interstitium,alveoli and pulmonary vessels,mainly neutrophils,lymphocytes,eosinophil cells and so on.2.Edu labeling bone marrow mesenchymal stem cells autologous reinfusion and 4weeks,8 weeks after antioxidative treatment had effects on rabbit emphysema model.Before the infusion of bone marrow mesenchymal stem cells,the inflammatory factor concentrations in each group were almost at the same level(P>0.05).There was a statistically significant in difference between the plasma IL-10 and TGF-?groups(P<0.05)after stem cell infusion.There was no significant in difference between model control group and antioxidant group(P>0.05),low-dose stem cell treatment group and low dose stem cells with anti-oxidation treatment group(P>0.05),high-dose stem cell treatment group and high-dose stem cells combined with anti-oxidation treatment group(P>0.05).Plasma IL-10and TGF-?increased as the observation time prolonged between the other groups(P<0.05)and at different time points(P<0.05).The concentration in IL-10 and TGF-?in alveolar lavage fluid increased obviously as the greater the number of stem cell infusions(P<0.05).Comparing plasma levels of TNF-?,H2O2,IL-1(P<0.05),there was no significant in difference between model control group and antioxidant group(P>0.05),low-dose stem cell treatment group and low dose stem cells with anti-oxidation treatment group(P>0.05),high-dose stem cell treatment group and high-dose stem cells combined with anti-oxidation treatment group(P>0.05)after stem cell infusion.Plasma TNF-?,H2O2,and IL-1decreased as the observation time prolonged between the other groups(P<0.05)and at different time points(P<0.05).The concentration in TNF-?,H2O2,and IL-1 in alveolar lavage fluid decreased obviously as the greater the number of stem cell infusions(P<0.05).There were no significant differences in heart rate,respiratory rate and blood oxygen saturation between the groups before and after bone marrow mesenchymal stem cell transfusion(P>0.05).They were compared at different time points(P>0.05).It showed HE staining of lung tissues in each group.Severe destruction of lung tissue in model control group and antioxidant group,ruptured lung septa,alveolar fusion,pulmonary interstitial hyperplasia,massive infiltration of inflammatory cells,alveolar inflammation was recuded in the stem cell treatment group,and alveolar monolayer epithelium cells were arranged.Treatment with high-dose stem cells and high-dose stem cells combined with anti-oxidation treatment was appeared to be better.There was no Edu positive cells when lung tissue fluorescence staining were observed under fluorescent microscope.Conclusions1.It successfully constructed a COPD animal model by using trypsin nebulization;2.It was successful by using the isolation,culture,amplification and marking of BM-MSCs;3.By injecting BM-MSCs into the COPD model rabbits via ear vein autograft,the more the amount of stem cells transfused,the more pronounced the reduction of pro-inflammatory cytokines IL-1,TNF-?,and H2O2 in plasma and bronchoalveolar lavage fluid,and also the more significant factor TGF-?and IL-10 increased.4.Antioxidant therapy has little effect;5.In lung tissue HE staining,inflammatory cells in the BM-MSC treatment-related group were significantly reduced.Tthe lung tissue structure was improved compared with the model control group.With the prolongation of stem cell transfusion,the average lung volume gradually decreased,indicating that the bone marrow mesenchyme stem cells have a certain repair effect on COPD;6.Edu positive cells were not detected by fluorescent staining of lung tissue;... |
PDF Full Text Request