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《Chinese Journal of Reparative and Reconstructive Surgery》 2015-01
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CONDITIONS OF SYNOVIAL MESENCHYMAL STEM CELLS DIFFERENTIATING INTO FIBROCARTILAGE CELLS

FU Peiliang;CONG Ruijun;CHEN Song;ZHANG Lei;DING Zheru;ZHOU Qi;LI Lintao;XU Zhenyu;WU Yuli;WU Haishan;Department of Joint Surgery,Shanghai Changzheng Hospital;  
Objective To explore the conditions of synovial derived mesenchymal stem cells(SMSCs) differentiating into the fibrocartilage cells by using the orthogonal experiment. Methods The synovium was harvested from 5 adult New Zealand white rabbits,and SMSCs were separated by adherence method. The flow cytometry and multidirectional differentiation method were used to identify the SMSCs. The conditions were found from the preliminary experiment and literature review. The missing test was carried out to screen the conditions and then 12 conditions were used for the orthogonal experiment,including transforming growth factor β 1(TGF-β1),bone morphogenic protein 2(BMP-2),dexamethasone(DEX),proline,ascorbic acid(ASA),pyruvic acid,insulin+transferrin+selenious acid pre-mixed solution(ITS),bovin serum albumin(BSA),basic fibroblast growth factor(b FGF),intermittent hydraulic pressure(IHP),bone morphogenic protein 7(BMP-7),and insulin-like growth factor(IGF). The L60(212) orthogonal experiment was designed using the SPSS 18.0 with 2 level conditions and the cells were induced to differentiate on the small intestinal submucosa(SIS)-3D scaffold. The CD151+/CD44+ cells were detected with the flow cytometry and then the differentiation rate was recorded. The immumohistochemical staining,cellular morphology,toluidine blue staining,and semi-quantitative RT-PCR examination for the gene expressions of sex determining region Y(SRY)-box 9 gene(Sox9),aggrecan gene(AGN),collagen type I gene(Col I),collagen type II gene(Col II),collagen type IX gene(Col IX) were used for result confirmation. The differentiation rate was calculated as the product of CD151/CD44+ cells and cells with Col I high expression. The grow curve was detected with the DNA abundance using the Pico Green Assay. The visual observation and the variances analysis among the variable were used to evaluate the result of the orthogonal experiment,1 level interaction was considered. The q-test and the least significant difference(LDS) were used for the variance analysis with a type III calibration model. The test criteria(α) was 0.05. Results The cells were certified as SMSCs,the doubletime of the cells was 28 hours. During the differentiation into the fibrocartilage,the volume of the SIS-3D scaffold enlarged double every 5 days. The scaffolds were positively stained by toluidine blue at 14 days. The visual observation showed that high levels of TGF-β1 and BMP-7 were optimum for the differentiation,and BMP-7 showed the interaction with BMP-2. The conditions of DEX,ASA,ITS,transferrin,b FGF showed decreasing promotional function by degrees,and the model showed the perfect relevance. P value was 0.000 according to the variance analysis. The intercept analysis showed different independent variables brought about variant contribution; the TGF-β1,ASA,b FGF,IGF,and BMP-7 were more remarkable,which were similar to the visual observation. Conclusion In the process of the SMSCs differentiation into the fibrocartilage,the concentrations of TGF-β1,ASA,b FGF,and IGF reasonably can improve the conversion rate of the fibrocartilage cells. The accurate conditions of the regulatory factor should be explored further.
【Fund】: 国家自然科学基金青年基金资助项目(81000798);; 上海市科委自然科学基金(15ZR1414000)
【CateGory Index】: R329.2
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