Inducing The Rat Bone Mesenchymal Stem Cell To Express Chondrogenic Phenotype By Up-regulation Expressing ChM-Ⅰ

Objective:To study the role of the ChM-Ⅰin up-regulated inducing BMSCs intochondrocytes.Methods:1. MSCs were obtained from SD rats using the method of density gradientcentrifugation and subculturing. Cell surface antigens(CD90、CD29) were detected byflow cytometry.The frozen MSCs were transfected with the plasmid pcDNA3.1(+)/ChM-Ⅰ and transfected with the plasmid pcDNA3.1(+), which were recoveriedand brought up.2.Serumfree chondroinductive medium contained10ng/ml TGF-β1、100nmol/Ldexamethasone、50umol/ml ascorbic Acid and1％ITS.3. Six groups were divided: experimental group1(stable transfected with theexpression vector pcDNA3.1(+)/ChM-ⅠMSCs group)；experimental group2(stablytransfected with expression vector pcDNA3.1(+)/ChM-ⅠMSCs group and additinginductionfluid)；control group1(normal MSCs group)； control group2(normal MSCsand induced fluid group)；control group3(stably transfected with the expression vectorpcDNA3.1(＋)MSCs group)；control group4(stably transfected with the expressionvector pcDNA3.1(＋)MSCs and induced fluid group)，the cells cultured for7days、14days、21days, using the inverted microscope observation of morphological changes inthe characteristics；toluidine blue staining of cartilage the secretion of matrix；immunocytochemical detection of cartilage-specific typeⅡcollagen expression.Through the above experimental methods, identification shows that BMSCs cell lineswith the expression of ChM-Ⅰcapable of differentiation to chondrocytes.Result:1. amplification and cultivation of MSCs cell line The medium was changed after48h the single-cell suspension. Using invertedmicroscope and photographing, you can see the spindle-shaped or triangular adherentcells at the bottom of the Petri dish. The cell density reached more than85%aftercultured11~13days. Through being passaged, more than80%cells were adherent in6hours; cells grown accelerated like fibroblast; strong refraction; community like shoal.Flow cytometry testing results: MSCs positive for surface antigens of CD90、CD29expression rate were99.79%,98.41%; MSCs negative surface antigens of CD45、CD11b/c expression rates were2.77%,2.51%. Indication shows that MSCs are to meetexperimental requirements.2. Resuscitation and cultivation of MSCs cell line―the MSCs transfectedwith the plasmid pcDNA3.1(+)/ChM-Ⅰand the MSCs transfected with the plasmidpcDNA3.1(+)After resuscitating, through cultivation and passage, the cells (the MSCstransfected with the plasmid pcDNA3.1(+)/ChM-Ⅰand the MSCs transfected with theplasmid pcDNA3.1(+)) growth vigorous, were densely woven-like or spiral-shapedneatly arranged.3. The results and analysis of toluidine blue and immunohistochemistrystaining of typeⅡcollagen at the six groupsThe cells (induced experimental group2and the3control groups) changed from aspindle-like fibroblastic appearance to a polygonal shape. Toluidine blue staining andtypeⅡcollagen immunohistochemical results showed that：the testing results (thespecific secretion of cartilage matrix and the expression of cartilage typeⅡcollagen) ofcontrol group1、3were all negative; experimental group1and control group2、4weredetected in the presence of the specific secretion of cartilage matrix and the expressionof cartilage typeⅡcollagen, but the experimental group1was weakly positive, controlgroup2、4were positive, indicating the up-regulation expression of ChM-Ⅰcan induceMSCs to chondrocyte differentiation, but less than the efficiency of the TGF-β1; thetesting results (the specific secretion of cartilage matrix and the expression of cartilagetypeⅡcollagen) of experimental group2were strongly positive, better than the control2、4；experimental group2and group1could be detected in specific cartilage matrixsecretion and cartilage typeⅡcollagen expression, and experimental group2was betterthan the experimental group1, indicating that the MSCs（stable transfected with theexpression vector pcDNA3.1(+)/ChM-Ⅰ）can significantly increase the ability todifferentiate to chondrocytes in the synergistic effect of TGF-β1. Conclusion:1.The experiment proved successfully that ChM-Ⅰcan induce differentiation ofMSCs into chondrocytes.2.Upregulated ChM-Ⅰ MSCs can increase the ability to differentiate tochondrocytes in the synergistic effect of TGF-β1.3.The mode of combining to induce MSCs by multiple expression vectors and avariety of biological factors may become an important way for cartilage tissueengineering.