| Part 1 Isolation, culture, identification and labeling of human umbilical cord-derived mesenchymal stem cellOBJECTIVE:To explore the methods of isolation, cultivation, identification and fluorescent-labeling human umbilical cord mesenchymal stem cells (hUC-MSCs) and study its biological characteristics, prepared for the late-stage experiments in vitro and in vivo.METHODS:HUC-MSCs were cultured with enzymolytic method and tissue block method respectively, in DMEM/F12 medium containing 10% FBS, with 5% CO2 in air at 37 ℃ and were subcultured at the ratio of 1:3 every 3-4 d. The morphological changes were observed by the inverted microscope, the time of beginning harvest original generation (PO) cells and output of PO cells were statistics, cell proliferation capacity was detected by CCK8 kit, living cells ratio of cryopreserved recovery was detected by trypan blue staining. Through observation of cell adherent capacity, cell phenotype detected by flow cytometry, differentiation capacity of adipogenic, osteogenic, chondrogenic in vitro, whether the mesenchymal stem cells meet international standards was identified. Adenovirus vector carrying green fluorescent protein gene (Ad-GFP) transfected hUC-MSCs for cell labeling, transfection efficiency was detected by flow cytometry.RESULTS:Morphology of hUC-MSCs was spindle-shaped appearance and grew in radially or whirl manner in the hypercellular areas. The PO cells of enzymolytic method harvesting time earlier than tissue block method (7.75±2.50 d VS 11.00 ± 0.82 d, P< 0.05), but output is lower than the latter (1±0.24×105/cm VS 1±0.42×106/cm, P<0.05).Cell proliferation, living cells ratio of cryopreserved recovery has no obvious difference for two methods. Cell obtained by two methods had the adherent growth ability, the positive ratio detected by flow cytometry were CD45 (0.437%), CD90 (96.7%), CD105 (34.9%), CD146 (92.4%). In vitro induced environment, hUC-MSCs had the adipogenic, osteogenic, chondrogenic differentiation ability. HUC-MSCs transfected by Ad-GFP shows strong green fluorescence, GFP positive rate detected by flow cytometry was 94.4%.CONCLUSIONS:Methods of enzymolytic method and tissue block method can produce enough PO hUC-MSCs. Through observation of cell adherent capacity, cell phenotype detected by flow cytometry, differentiation capacity of adipogenic, osteogenic, chondrogenic in vitro, hUC-MSCs meet international standards. HUC-MSCs transfected by Ad-GFP have high efficiency, strong fluorescence characteristics.Part 2 Effect of hUC-MSCs on repairing the guinea pigs nasal mucosa radioactive damageOBJECTIVE:To test the therapeutic effect of hUC-MSCs intravenously injection and conditioned medium nasal inhalation on guinea pig nasal mucosa radiation damage model.METHODS:Constructing guinea pig nasal mucosa radiation damage model, treated with hUC-MSCs intravenously injection or concentrated conditioned medium nasal inhalation. To evaluate therapeutic effect,1 week,1 month,3 month and 6 month after radiotherapy, mucuscilia clearance time (MCT) was detected by charcoal-powder method, nasal mucosa pathologic change was detected by nasal mucosa biopsy HE staining, mucosa cilia form and coverage ratio was detected by scanning electron microscopy, normal guinea pigs and radiation therapy but not to interfere in guinea pig as a control.RESULTS:The mucuscilia clearance time, degree of mucosal edema of hUC-MSCs intravenously injection and concentrated conditioned medium nasal inhalation groups at 1 week and 1 month were better than control group, but no difference was found at 3,6 months. The cilia coverage of concentrated conditioned medium nasal inhalation group at 6 month was better than control group.CONCLUSIONS:hUC-MSCs intravenously injection or concentrated conditioned medium nasal inhalation have a therapeutic effect on guinea pig nasal mucosa radiation damage model.Part 3 The mechanism study of hUC-MSCs repairing nasal mucosa radiation damageOBJECTIVE:To approach the mechanism of hUC-MSCs repairing nasal mucosa radiation damage.METHODS:HUC-MSCs labeled with adenovirus vectors carrying green fluorescent protein gene (Ad-GFP) were injected intravenously into the guinea pig model, the nasal mucosa specimens were detected by frozen section frozen section at 3 d,10 d,20 d,30 d after injection, GFP marked cells migration, differentiation were detected by directly observing under fluorescence microscope and immunofluorescence stain method. Nasal mucosa epithelial cells were cultured and were identified in vitro, then co-culture with hUC-MSCs. Observing hUC-MSCs morphological changes, whether it express the nasal mucosa epithelial cells specificity protein, chemotaxis to nasal mucosa epithelial cells after radiation and influence of hUC-MSCs conditioned medium on nasal mucosa epithelial cell proliferation, with the purpose of approach the mechanism of hUC-MSCs repairing nasal mucosa radiation damage.RESULTS:Ad-GFP labeled hUC-MSCs can be observed directly by frozen section with fluorescence microscope in 3 and 10 days after injection and can be detected by immunofluorescence staining in 20 days after injection, which mainly located in the mucosa lamina propria and cannot express the β-Tubulin protein. When hUC-MSCs were co-culture in vitro with human nasal mucosa epithelial cells, some hUC-MSCs morphology were change and can express epithelial cell specific protein keratin. HUC-MSCs has chemotaxis to the radiated nasal mucosa epithelial cells and hUC-MSCs conditioned medium can promote nasal mucosal epithelial cell proliferation.CONCLUSIONS:HUC-MSCs can promote mucus cilia system function recovery of guinea pigs nasal mucosa radiation model by paracrine mechanism. |
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