Study On The Engraftment And Differentiation Of Bone Marrow Mesenchymal Stem Cells By Overexpressing Wnt3a In The Lung Tissue Of Rat Models With Chronic Obstructive Pulmonary Disease

Objective:To explore the impact of Wnt3 a overexpression on the engraftment rate and differentiation rate of bone marrow mesenchymal stem cells in the lungs of rat models with chronic obstructive pulmonary disease(COPD),and to preliminarily study the role of Wnt3 a in the treatment of COPD.Method:1.Cultivation and identification of bone marrow-derived mesenchymal stem cells(BMSCs): Rat MSCs were acquired with bone marrow adherence method and then labeled with CM-Dil after phenotypic identification by flow cytometry.2.Transfect P3 rat BMSCs with Wnt3 a lentivirus and evaluate the Wnt3 a transfection efficiency with western and qPCR method.3.The COPD rat models were developed in one month by the injection of lipopolysaccharide into the airway coupled with smoking.The treatment of each group was presented as follows: normal control group(group A): normal rats with the injection of 1 ml PBS via tail vein;COPD group(group B): COPD rat models with 1 ml PBS injected via tail vein;BMSCs group(group C): COPD rat models with P3 BMSCs labeled with CM-Dil injected via tail vein;Wnt3a-BMSCs group(group D): COPD rat models with Wnt3a-overexpressed P3 BMSCs labeled with CM-Dil injected via tail vein.After modeling,the rats in each group were executed in batches on day 7,day 14,and day 28,and each lung tissue section was gathered and then stained with HE to observe the pathological changes.4.Analyze the engraftment and differentiation of BMSCs in lung tissue: a)Observe the lung tissue section under a laser confocal microscope,count CM-Dil-labeled cells and all cells in the same field with Image J software,ending up with the analysis of the BMSCs engraftment rate in the lung tissue rate.b)stain lung tissue sections with immunofluorescence and count BMSCs expressing alveolar epithelial specific protein SPC,CM-Dil labeled BMSCs,and all cells in the same field.Image J software was employed to count double-positive cells,ending up with counting the differentiation rate of BMSCs in COPD lung tissue.Result:1.Flow cytometry discovered the high expressions of the surface markers containing CD29(97.4%)and CD44(74.6%)and the low-expression of CD34(0.13%)and CD45(0.56%)in rat BMSCs.2.The over-expression of Wnt3 a in BMSCs transfected with lentivirus was detected with Western and qPCR method.3.HE staining showed that the alveolar structure of the COPD group(group B,C,D)was destroyed when compared with that in normal control group(group A),indicating that the modeling was successful.4.Under a laser microscope,there were CM-Dil(red fluorescent)labeled BMSCs in the lung tissue slices in BMSCs group(group C)and Wnt3a-BMSCs group(group D),whilst the colonization rate of BMSCs in Wnt3a-BMSCs group(group D)was higher than that in BMSCs group(group C)(P< 0.05),demonstrating that the overexpression of Wnt3 a enhanced the engraftment of BMSCs in lung tissues.The immunotissue fluorescence staining found that CM-Dil labeled BMSCs can simultaneously express alveolar epithelial cell specific protein SPC(green fluorescence)in BMSCs group(group C)and Wnt3a-BMSCs group(group D),while the proportion of double-positive cells in Wnt3a-BMSCs group(group D)was significantly higher than that in BMSCs group(group C)(P<0.05),suggesting that overexpression of Wnt3 a can promote the differentiation of BMSCs in the lung.Conclusion:Injection of BMSCs overexpressing Wnt3 a into COPD rat models can promote the engraftment of BMSCs in lung tissue and the differentiation of BMSCs into alveolar epithelial cells,thus improving the pathological structure of damaged lung tissue and promoting the process of lung repair.