BMP-2in Combination With IGF-Ⅰin Witro Inguced Bone Marrow Mesenchymal Stem Cells Into Cartilage Cells In Expermenal Study

PART ⅠThe separation， culture and identification of bonemarrow mesenchymal stem cellObjective To master the technique of primary and passage culture ofbone marrow mesenchymal stem cells，To lay the foundation for subsequentexperimentMethods Using the whole bone marrow culture method to isolatedand cultured Wistar rat bone marrow-mesenchymal stem cells(BMSC)． Using immunohistochemical staining and flow cytometry toidentify the third generation of bone marrow mesenchymal stem cells, todetect the expression of cell surface antigens CD34, CD44and CD90，using MTT assay to detect proliferation of bone marrow mesenchymal stemcell and to make cell growth curve mapping.Results Bone marrow mesenchymal stem cells grow adherently and colonily in vitro, cells were spindle-shaped, polygonal, and had differentsizes of Protrusions， contained the larger particles in the cytoplasm, thenuclear oval, cell division relative to more common, the cells growth curveshowing cells grow in good condition. the identification results of the thirdgeneration of mesenchymal stem cells (BMSC)was that expression of CD44antigen positive, CD90positive rate95.5%, CD34-positive rate0.21%,Comply with the requirement of experiment.Conclusion Bone marrow mesenchymal stem cells readily available,source sufficient, can be greatly amplified by passage, After accession toinducing medium Cell shape changes may be obtained from cartilage cells,Can be used as seed cells for cartilage tissue engineering. PART ⅡGFP gene transfection,induction of bone marrowmesenchymal stem cells into cartilage cells in experimental studyObjective Use of green fluorescent protein (GFP) marker marktransfected bone marrow mesenchymal stem cells (BMSCs)；To discussionthe ability of Rat bone marrow mesenchymal stem cells (BMSC) in vitroinduced by different growth factors to induce into cartilage cells． Toestablish an ideal chondrocytes induced conditioned medium, in order toprovide seed cells in cartilage repair, to provide experimental theoryevidence for artilage tissue engineering.Methods To culture ang passage in vitro, Taking the third-generationBMSC and using the culture medium which containing BMP-2(bonemorphogenetic protein-2), and IGF-Ⅰ (insulin-like growthfactor-1)-induced factors in monolayer cell culture model to observe theability of cartilage cells were differentiated, The experiments were dividedinto GFP transfection group and non-transfected group. GFP transfectedgroup was divided into four groups: BMP-2(100ng/ml)-induced group,IGF-Ⅰ (10ng/ml)-induced group, IGF-Ⅰ, BMP-2(100ng/ml,10ng/ml)Co-induction group, did not add any growth factor group；Non-transfectedgroup also divided into four groups: BMP-2(100ng/ml)-induced group,IGF-Ⅰ (10ng/ml)-induced group, IGF-Ⅰ, BMP-2(100ng/ml,10ng/ml) Co-induction group, did not add any growth factor group. Usingimmunohistochemical staining and flow cytometry to identify the thirdgeneration of bone marrow mesenchymal stem cells, to detect the expressionof cell surface antigens CD34, CD44and CD90， using MTT assay todetect proliferation of bone marrow mesenchymal stem cell and to make cellgrowth curve mapping，Induced14days，to observe cells in general byinverted microscope， to observe fluorescence by fluorescence microscopyfor GFP transfected group. With Alcian staining to detect the secretion ofglycosaminoglycan， to detect the expression of collagen type Ⅱ byimmunohistochemistry.To use dimethyl sulfoxide methylene blue (DMB)colorimetric quantitative detection thesecretion of glycosaminoglycan(GAG).Results Alcian staining and type Ⅱ collagen immunohistochemistrywere positive in the induction group， And glycosaminoglycan (GAG)secretion of IGF-Ⅰ, BMP-2induced by the combined group was higher thanother groups in the same group（P<0.05），However, transfection group andnon-transfected group, IGF-Ⅰ, BMP-2induced by the combined group ofglycosaminoglycan (GAG) secretion in the amount of no significantdifference （P<0.05）. Transfected with GFP group at24hours aftertransfection,1week,2weeks,3weeks,4weeks, green fluorescent can beexpressed underthe fluorescence microscope，and the GFP gene transfectionhad no significant impact on BMSCs proliferation and differentiation. Conclusion： IGF-Ⅰ and BMP-2can induce bone marrowmesenchymal stem cells the direction of the chondrocyte differentiation, InDMEM medium by adding IGF-Ⅰ and BMP-2can be used as an idealconditioned medium of chondrocytes induced. GFP gene into bone marrowmesenchymal stem cells (BMSC) which activity and biological charact-eristics have no significant impact，and the green fluorescence could be along-term expression in cells，Green fluorescent protein (GFP) markertechnique can be used as an ideal tissue-engineered cell marker tracertechnique.