Research Of Culture, Identification And Biological Characteristics Of Human Olfactory Mucosa Mesenchymal Stem Cells

Chapter one: Obtain,culture,and identification of human olfactory mucosa mesenchymal stem cellsObjective: To establish an easy and feasible method of obtainment,isolation and cultivation of human OM-MSCs in vitro.Methods: Through the olfactory mucosa adherent method,olfactory mucosa mesenchymal stem cells were isolated,cultured in vitro.Morphological observation was observed by microscope.Surface markers were detected by flow eytomeyrty instrument.The cells were induced towards adipocyte,osteocyte.Following induction,Lipid droplets showed red color after oil red O staining and alizarin red staining produced deposit of calcium in cells.By immunofluorescence experiments,the olfactory mucosa mesenchymal stem cell surface markers Nestin,STRO-1 was observed whether they are positive expressed.Results: OM-MSCs from human were spindle cell-based,showing radial colony arrangement.Cells kept strong growth.At the5 passage,OM-MSCs were negative for CD34 and CD45,but positive for CD73,CD90 and CD105.Moreover OM-MSCs were capable of adipogenic and osteogenic.Following immunofluorescence,the olfactory mucosa mesenchymal stem cell surface markers Nestin and STRO-1 were positive expressed.Conclusion: Human olfactory mucosa tissue adherence method is easy and can isolate and amplify OM-MSCs in vitro.This experimental method has important practical significance to provide adequate source of seed cells for clinical application.Chapter two: The biological characteristics of human olfactory mucosa mesenchymal stem cellsObjective: The purpose of this study was to observe the biological characteristics of the human olfactory mucosa mesenchymal stem cellsMethods: 1 According to the method of the part one that the OM-MSCs were isolated,purified,cultured and passaged.2Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of human OM-MSCs.3 Add neural stem cells medium(DMEM/F12 containing 2%B27+ 20μg/L epidermal growth factor+ 20μg/L basic fibroblast growth factor)to observe the olfactory mucosa between mesenchymal stem cells forming into sphere.4 Growth curve was measured by MTT.5 Cell cycle analysis was test by flow cytometry instrument.6 The cells were induced towards neural-like-cells in vitro.Immunocytochemistry and western blot were used to detect the expression ofβ-tubulin,GFAP and MAP-2.SEM and patch clamp are used for observation the ultrastructure of neurons and neurons electrophysiological properties respectively.Results: The short and thick microvilli processes were seen at the surface of OM-MSCs by scanning electron microscope.2kind of cellular morphology of OM-MSCs were seen under transmission electron microscope and OM-MSCs can form spheres.The growth curve demonstrated that OM-MSCs were consistent with the growth characteristics and good activity of normal cells.Cell cycle analysis showed that more than 80%MSCs were at G0-G1 phase.After induction of neural differentiation,OM-MSCs high express neuronal markers MAP-2and tubulin,with nissl body and potassium channels.Conclusion: It is concluded that the cells isolated and cultured from human olfactory mucosa possess biological characteristics of MSCs and display multiple differentiation.They can be utilized as ideal seed cells to repair tissue-engineering.