| Backgrounds and Objectives: To explore the best cytokine combination of inducingsynovial mesenchymal stem cells (SMSCs) differentiating into cartilage. On this basis,exploring the microRNAs (miRNAs) expression spectrum during differentiation process,and discussing the effect of miRNA on the cartilage differentiation by building the miRNAover expression system.Methods:1.According to Pei’s cultivation methods, SMSCs were isolated from the kneejoints synovial tissue of New Zealand white rabbit, and the harvested MSCs wereidentified by morphological observation, flow cytometry, adipogenic, osteogenicdifferentiation.2.To explore the optimal conditions, the PELLET cultivation system wereused, and the polypropylene pipe were divided into8groups with added transforminggrowth factor-β3(TGF-β3), bone morphogenetic protein-2(BMP-2), and dexamethasone(DXM) independently, binary, or trinary. The diameter, weight, collagen II andproteoglycan of cell pellets of each group were comparied to evaluat the effect of cytokineson the chondrogenic differentiation of SMSCs. By comparingthe diameter, weight,cartilage proteoglycan (toluidine blue staining), collagen type II (immunohistochemicalstaining) expression, and cartilage marker gene expression level (quantitative rt-pcr) toevaluate each cytokine ability of inducing SMSCs differentiate into cartilage, and finallydetermineing the optimal cytokine combination.3.Third-generation SMSCs were dividedinto two groups: experimental group added with TGF-β3, BMP-2and DXM, the controlgroup without these cytokines. At7d,14d and21d of the induction process, the expressionof miRNAs were detected by microarray gene chip technology analysis, and the RT-qPCRtechnology to authenticate the differentially expressed miRNAs. On the basis of theexperimental group, SMSCs were divided into A, B two groups: group A as theexperimental group, transfection miR-218mimics the expression, group B as controlgroup, transfection mimics-NC, and then verified the effect of over expression miR-218onthe SMSCs’capability of differentiating into cartilage.Results:1.Flow cytometry showed that the specific surface antigen CD44, CD106, CD105and CD90are positive expression, oil red O staining and alkaline phosphatase (ALP)staining results are also positive, those indicate that the isolated stem cells have the specificphenotypeof mesenchymal stem cells and have the multidirectional differentiationpotential.2.Under the condition of different combination of cytokine inducing SMSCsdifferentiate into cartilage, the cell pellets induced with TGF-β3, BMP-2and DXM have the maximum diameter, the weight of the heaviest, and amount of proteoglycan andcollagen type II synthesis is also the most; Quantitative rt-pcr results also showed that therelated gene expression level is most highest under this condition (P＜0.01).3.Microarraygene chip technology screen out more than16miRNAs show differences in2times in theprocess of differentiation compared with control group. Among them, nine miRNAs wereupregulated and seven were downregulated. RT-qPCR method is used to verified theupregulation expression miR-340/140and downregulation miR-21/132, the results showthe RT-qPCR are consistent with the results of the microarray chip, this chip study resultsare true and correct. Experimental were transfected miR-218mimics to over express it, theresults showed that the cartilage specific cytoplasm component GAG was significantlyreduced compared with control group.Conclusion:1.The stem cells isolated according to Pei’s methods have the specificphenotypeof mesenchymal stem cells and have the multidirectional differentiationpotential.2.The cytokines combination of TGF-β3, BMP-2and DXM can make thecapacity of chondrogenic differentiation of SMSCs maximized.3.Many miRNAs wereinvolved in regulating the differentiation process of SMSCs differentiate into cartilage;Over the expression of miR-218can significantly inhibit the capability of chondrogenesisof SMSCs. |
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