Regulation Of Wnt Pathway On Adipose Derived Stem Cells Into Cartilage Differentiation

Part I Isolation,purification and culture of adipose stem cellsObjective:To establish a simple and effective method for isolation culture of SD rat adipose-derived stem cells,and to provide an ideal seed cell for tissue engineering.Methods: 1.Healthy adult male Sprague-Dawley rats were used to cut off the subcutaneous fat in the abdomen and groin.Adipose-derived stem cells were isolated by enzymatic digestion.The adipose-derived stem cells were obtained by in vitro culture,continuous passage and adherent purification.2.The morphological changes of adipose stem cells were observed by inverted phase contrast microscopy.The expression of surface antigen CD44 was detected by immunofluorescence.3.The morphological changes of adipose-derived stem cells were observed by inverted phase-contrast microscope.The adipose-derived stem cells were isolated and cultured into cartilage,osteogenic differentiation,specific reagents for cell staining to determine the differentiation potential of adipose-derived stem cells.Results: 1.After 4h the cells can be seen adherent,scattered distribution,adherent cells after 48 h tentacles reach out,the volume increases,was fibroblast-like short fusiform,particles gradually increased,five or six days after the cells were colony-like growth,Cells become long fusiform,spiral arrangement,after passage,the cell morphology is uniform,the formation of clones.2.Immunofluorescence assay showed red fluorescence,Dapi stained nuclei were blue,ADSCs cell surface antigen CD44 was positive expression.3.Adipose-derived stem cells differentiated into chondrocytes to form densely packed cell mass,and then stained with alitame blue,a large number of blue matrix particles were observed in the cells under microscope.After alveolar red-staining,alizarin red staining showed a lot of orange-red Calcium nodules.Conclusion: Isolation by enzyme digestion,adherent purification of desalinated adipose-derived stem cells,good growth,rapid proliferation,high purity,is a simple and effective method of sorting and culture,can be an ideal seed cells for the follow-up experiments to provide reliable The stable cell source.Part 2 Wnt pathway and Sox9 feedback each other to regulate adipose derived stem cells into cartilage differentiation roleObjective:To investigate the effect of Wnt pathway on the ability of adipose derived stem cells to differentiate into chondrogenic cells,and to observe the expression of Sox9 during adipogenic differentiation of adipose derived stem cells into chondrocytes.The study focused on the relationship between Wnt pathway and adipose-derived stem cells in regulating chondrogenic differentiation.If it can be applied to clinical,it is believed that it can provide experimental basis for the repair and regeneration of articular cartilage tissue defects in bone and joint,which is of great significance to the further and extensive application of ADSCs in the field of rehabilitation of bone and joint.Methods: 1.After stimulating stimulated adipose stem cells with agonist Li CL,the proliferation activity of ADSCs was observed by CCK-8 method and the expression of PCNA protein was detected by Western blot.2.Adipose stem cells of chondrogenic differentiation of each period,cartilage index Sox9,Aggrecan,Collagen2 a Wnt pathway and a key molecule ?-catenin for Western blot,RT-q PCR detection level,to assess their ability to differentiate into chondrocytes.3.Li CL agonist,inhibitor Dkk-1 stimulation intervention ADSCs chondrogenic differentiation,7 days after the intervention focus detection(early),day 21(late),cartilage index Sox9,Aggrecan,Collagen2 a Wnt pathway and Key Molecular ?-catenin protein and m RNA expression.4.Adipose stem cells into chondrogenic differentiation day 21-day 35,RT-q PCR detection of m RNA levels of mature chondrocytes Collagen2 a,Collagen10 and osteogenic differentiation indicators RUNX2 early,Western blot detection Sox9,?-catenin protein Express the situation.5.Li CL agonist,inhibitor Dkk-1 stimulation intervention ADSCs chondrogenic differentiation day 28(late),Western blot detection Sox9,?-catenin protein levels and Collagen2 a,RUNX2 Differentiation of protein.6.As the use of lentiviral vectors,the Sox9 gene transfer into adipose stem cells to obtain stable expression,RT-q PCR detected during chondrogenic differentiation specific markers Collagen2 a,Aggrecan levels of expression,quantitative determination of secreted glycosaminoglycans content.Western blot was also performed on Sox9 and beta-catenin to assess their ability to differentiate into cartilage.7.Sox9 lentiviral vector positively transfected adipose stem cells on day 21,the expression immunofluorescence Collagen2 a cartilage differentiation induction,Western blot detection of Wnt pathway proteins ?-catenin,,expression of p-?-catenin protein GSK-3?;Results:1.Stimulated by Li CL stimulated ADSCs,ADSCs proliferation increased,and PCNA protein showed high expression.2.The m RNA and protein expression of Sox9,Aggrecan and Collagen2 a in cartilage showed an increasing trend over time.The content of GAG increased also and increased on the 14 th day Significant.The Wnt pathway key molecule ?-catenin protein is reduced and then increased.3.On the 7th day(early)and the 21 st day(late),Li CL promoted the expression of Sox9,Aggrecan,Collagen2 a,and ?-catenin,while Dkk-1 inhibits the expression of all cartilage markers.4.The expression of Collagen2 a decreased gradually with the passage of time.The m RNA expressions of RUNX2 and Collagen10 gradually increased,the expression of ?-catenin increased and the expression of Sox9 decreased gradually.Li Cl promoted the expression of ?-catenin,and down-regulated the expressions of Sox9 and Collagen 2a,but the results of Dkk-1 were opposite.5.Sox9 lentiviral vector positive transfected adipose-derived stem cells,set to transfection group,the Collagen2 a,Aggrecan m RNA levels were significantly higher expression of secreted glycosamino glycanes was also significantly higher expression;Western blot detection,Sox9 lentiviral transfection group significantly low expression.6.Chondrogenic differentiation on the 21 st day(late),immunofluore scence assay showed Sox9 lentiviral transfection group was significantly higher expression of Collagen2 a.The protein level of GSK-3? and p-?-catenin increased by Western blot.Conclusion: 1.Li CL can activate the Wnt pathway key protein?-catenin,in promoting chondrogenic differentiation in the rapid proliferation of adipose-derived stem cells.2.Li CL can activate Wnt pathway key protein?-catenin,which can promote the proliferation and differentiation of cartilage induced by Sox9 in the early stage;continue to regulate Sox9 to promote chondrogenic differentiation in the late stage,and gradually decrease Sox9 in the late stage,weaken the mature cartilage phenotype Expression,so that cartilage becomes hypertrophy,early osteogenesis.Dkk-1 stimulation inhibits the Wnt pathway,with the opposite effect.3.Overexpression of Sox9 could inhibit the expression of ?-catenin protein and promote its degradation,maintaining cartilage-induced differentiation.