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The first chapter of mouse bone marrow mesenchymal stem cells (MSCs) in vitro amplification and identification Objective mouse bone marrow-derived mesenchymal stem cells (marrow mesenchymal stem cells MSCs) in vitro isolation, purification and identification methods. Methods by density gradient centrifugation combined with adherent cells was cultured mouse MSCs, observe the different cells and growth morphology, flow cytometry surface antigen CD34, CD45, CD105 and CD106 expression, and mouse MSCs for determination of purity. Results: The primary cultured mouse MSCs seeded 24 hours after only a few visible adherent cells after 72 hours the number of adherent cells gradually increased, showing a number of colony-forming, this time adherent cell morphology as long spindle. 2-6 days in cell culture cells ,7-12 days slower rapid cell proliferation, was \Cells cultured for about two weeks, close to 80% -90% confluence, the cells are more homogeneous. After passage, substantially all of the mice MSCs24 hour adherent spindle-shaped or star-like shape, subculture the cells no longer colony growth mode, and showed a uniform distribution of growth. Cell viability obtained after purification was 92.6 ± 1.8%. Flow cytometry showed that the third generation of MSCs in mice uniformity better than 90%; CD34, CD45 expression was negative, CD105, CD106 expression was positive. Conclusion: Density gradient centrifugation and adherent cell culture method can be successfully cultured MSCs, vitality and purity of the resulting mouse MSCs were higher, can be identified by flow cytometry vitro cultured mouse MSCs. Chapter lung tissue with bone marrow mesenchymal stem cells in vitro co-culture Objective To establish a non-contact lung tissue of non-contact with MSCs cultured in vitro experimental model to explore hyperoxia lung tissue in vitro differentiation of MSCs and on the impact of migration. Methods born one day newborn mice were placed in 60% oxygen concentration at feeding 21 days, the establishment of bronchopulmonary dysplasia (bronchopulmonary dysplasia BPD) model of lung injury. 21 days were sacrificed by cervical lung tissue removed under sterile conditions, grinding, mesh filter, trypsin digestion, lung tissue was prepared single cell suspension. Set of 3 groups, transwell chamber (PET membrane pore size of 0.4um) for the third generation of the lower chamber mouse MSCs, were randomly placed in the upper chamber in hyperoxia lung single cell suspension (experimental group A), normal single cell suspension of lung tissue (experimental group B) and the blank culture medium (control) were cultured in vitro non-contact. Scratches were cultured for 6 days to do experiments, transwell chamber invasion assay was observed in each group after co-culture MSCs migration changes. Co-culture 8 days for immunofluorescence staining, confocal microscopy observed under co-culture system lower chamber SP-C, AQP5 immunofluorescence expression. Coculture 8 days using real-time quantitative (Real-time) PCR, 2 - △ △ Ct method of quantitative analysis of MSCs co-culture system in the lower chamber of surfactant protein-C (SP-C), aquaporin 5 (AQP5), transforming growth factor β1 (TGF-β1) mRNA expression levels. Results 60% of inhaled oxygen 21 days mouse lung tissue biopsy showed: normal alveolar structure disappeared, the alveolar lumen diameter significantly expanded, reducing the number of alveoli, showing a large area of ??the alveolar fusion alveolar septal thickening, a large interstitial cell hyperplasia, BPD hyperoxia lung injury model was successfully established. Scratches were observed 12 hours after training, three groups were only a small number of cells crawling to scratch in 24 hours has increased the number of the three groups, but the increase was more pronounced experimental group A, 48 hours experimental group and the control group B the number of migrating cells significantly less than the experimental group A. Transwell chamber invasion assay: After 48 hours, the small chamber on the lower surface of the cell count, the experimental group A and group B or experimental group compared to the number of cells through the PET film were significantly different. Tip damage lung tissue were cultured MSCs can promote migration capabilities. Immunofluorescence were cultured for 8 days Results: Group A can see the blue fluorescent marker hoechst33342, AQP5 red fluorescence, SP-C green fluorescence; experimental group and the control group B are seen only hoechst33342 blue fluorescent marker, AQP5 red fluorescence, but not see the SP-C green fluorescence. Were cultured for 8 days Real-timePCR Results: SP-CmRNA expression in the experimental group A exists, but the experimental group and control group B there were no SP-CmRNA expression. AQP5mRNA, TGF-β1mRNA three groups were expressed, the experimental group A and the experimental group and the control group B AQP5, TGF-β1mRNA significantly increased expression levels (P lt; 0.01). However, the experimental group, B group and control group had no significant difference in expression levels (P gt; 0.05). Conclusion successfully established BPD hyperoxia-induced lung injury model, as well as non-contact co-culture in vitro induced MSCs to differentiate into alveolar epithelial cells in experimental models, and confirmed and damage lung tissue were cultured in vitro migration ability of MSCs enhanced lung tissue damage can of mouse MSCs induced to differentiate into alveolar type Ⅱ epithelial cells (alveolar epithelial type Ⅱ cell AEC Ⅱ)
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