| Part 1.In vitro culture,purification and identification of adipose stem cellsObjective:To establish a convenient and efficient system for isolation,extraction and culture of adipose mesenchymal stem cells(ADSCs)in vitro,which provides a reliable source of cells for the development of cartilage tissue engineering.Methods:1.Fresh SVF was isolated by trypsin digestion,and adipose stem cells were obtained by continuous culture and passage in vitro.2.The cell morphological changes of ADSCs were observed with an inverted microscope,and the surface molecular characteristics of ADSCs cells were identified by flow cytometry.3.The isolated and extracted P3 generation ADSCs were induced to differentiate into osteogenic,adipogenic and chondrogenic cells,which further confirmed that ADSCs have multi-directional differentiation potentialResults:1.After 6 hours of cell culture,cells adhered to the wall and were scattered,with a small amount of impurity cells suspended in them;after 3 days,the cells adhered firmly,the particles increased,and the cells grew in colonies;after 7 days,the cells were arranged in a spindle shape or a fish group.Cell morphology after passage is consistent.2.Flow cytometric identification of membrane surface antigens in adipose MSCs showed that,compared to controls,CD73,CD105 and CD90 were positively expressed with 96.2%,99.7% and 99.6%,respectively,while CD34 and HLA-DR expression were negative,with positive rates of 2.31%,1.15%,respectively3.After 2 weeks of osteogenic induction and differentiation of ADSCs,a large number of orange-red calcium nodules were seen by alizarin red staining;after 2 weeks of adipogenic induction,bright red lipid droplets in the cytoplasm were seen by oil red O staining;chondrogenesis After induction of differentiation for 3 weeks,a large number of blue matrix granules were seen after staining with Alicia blueConclusion:Trypsin digestion and continuous passage method can obtain purified adiposederived mesenchymal stem cells,which is a simple and efficient method for separation and culture.The adipose stem cells cultured by the method have good growth state,strong proliferation ability and strong multi-directional differentiation ability,which can provide cell sources for subsequent experiments,and are an ideal cartilage tissue engineering seed cells.Part 2.The role of Wnt signaling pathway in ICA-induced chondrogenic differentiation of adipose-derived mesenchymal stem cellsObjective:To explore the role of canonical Wnt-β catennin signaling pathway in the regulation of chondrogenic differentiation of ADSCs by IcariinMethods:1.The effect of various concentrations of Icariin on the proliferation and differentiation of ADSCs was measured by MTT to observe the proliferative activity and whether the cells produced toxic reactions.2.Icariin,Wnt signaling pathway inhibitor DKK-1 stimulated intervention of adipose mesenchymal stem cells into cartilage induced differentiation,and m RNA of cartilage indicators SOX9,Collagen 2a,Aggrecan were detected by RT-PCR after 21 days expression3.Icariin,Wnt signaling pathway inhibitor DKK-1 stimulated adipose mesenchymal stem cells into chondrogenic induced differentiation,and the protein expression of cartilage indicators SOX9,Collagen 2a,Aggrecan and β-catenin,a key molecule of Wnt signaling pathway,were detected by Western-blot after 21 days.Results:1.The proliferative capacity of ADSCs cells was enhanced by Icariin stimulation.2.Icariin promotes the expression of the cartilage genes SOX9,Collagen 2a,Aggrecan and Wnt in ADSCs.expression of β-catenin,a key molecule in the signaling pathway,and DKK-1 inhibited the chondrogenic effect of Icariin and the expression of β-catenin3.Icariin can partially counteract the effect of Wnt signaling pathway inhibitor DKK-1 on chondrogenic differentiation of adipose-derived mesenchymal stem cellsConclusion:1.ICA can promote the proliferation and differentiation of adipose stem cells2.WNT/β-catenin signaling pathway is involved in the regulation of ICA-induced chondrogenic differentiation of ADSCs... |
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