The Researching On Osteoblasts And Cartilage Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells

Objective To establish a method isolating and culturing rabbit bone marrow stromal cells(BMSCs), a favorable conditions inducing chondrogenic differentiation and make an artificial cartilaginous explants using the differentiated chondrocytes. Then to repair the defects of rabbit articular cartilage using this explants.Method Extract of rabbit bone marrow3-4mL, using the lymphocyte separation medium to obtain mononuclear cells by gradient centrifugation, primary culture and subculture in vitro. The following experiment adoption primary cells and the plummy cells after five passages.(1) Inducing transformation of BMSCs to the reticulodromous interstitial cell:adding dexamethasone.bFGF, and vitamin C into the culture medium in order to inducing the celltransformation.(2) Inducing transformation of BMSCs to the osteoblasts: adding dexamethasone, D-glycerophosphate sodium and vitamin C into the culture medium in order to inducing the celltransformation, and detect the differences of the activity of alkaline phosphatase.(3) Inducing transformation of BMSCs to the chondrocytes:Adding dexamethasone,50ug/ml vitamin C,10ng/mL bFGF into the culture medium of the plummy BMSCs after2-3passages for one week,then change l0ng/mL bFGF tolOng/mLTGF-p, and keep on culture for3weeks. Observing the morphology and character of the cells. Detecting the character of chondrocytes or the directional origin of chondrocytes of transformational BMSCs by histological and polymerase chain reaction.Result BMSCs are induced by dexamethasone. bFGFand vitamin C for4weeks and then transform into bone marrow mesh mesenchymal stem cell.BMSCs are induced by dexamethasone. D-phosphoric acid sodium glycerin,bFGFand vitamin C for3-4weeks and transform into osteoblasts, and alkaline phosphatase trial is positive.When BMSCs are induced by dexamethasone, vitamin C (50ug/mL) andbFGF(10ng/mL) for1weeks.bFGF is changed into TGF-β(10ng/mL) and BMSCs are cultured for3weeks.cell form is firstly spindle then becomes round or oval. Cell secretes abundant extracellular matrix which are positive by toluidine blue and V-G.PCR shows that the cell mainly expresses mRNA of type I collagen but transformed cells mainly expresses mRNA of type II collagenConclusion The isolated BMSCs are capable of multiple cell line transformation, BMSCs retains stem cell-like transforming activity after long-term culture in vitro.BMSCs can transformed into chondrocytes or directional origin of chondrocytes under the certain induced environment.