Gene Chips Select Differential Representing Genes Of Mesenchymal Stem Cells And Chondrocytes

Objective: To find out the up-regulated differential representing genes both in hyaline chondrocytes and elastic chondrocytes , the genes which were up-regulated in the hyaline chondrocytes group and at the same time down-regulated in the elastic chondrocytes group, and the genes which were up-regulated in the elastic chondrocytes group and at the same time down-regulated in the hyaline chondrocytes group through selecting the differential representing genes of rat Mesenchymal Stem Cells, hyaline chondrocytes and elastic chondrocytes. Then the key genes in inducing the differentiation from MSCs to chondrocytes were found, this would lay a solid foundation for selecting the new growth factors which were able to induce the differentiation from MSCs to chondrocytes.Methods:SD Rat MSCs were purified with the Density Gradient Centrifugation. SD Rat elastic chondrocytes were acquired with the enzyme digestion method. After getting the SD rat costal cartilage and dissecting the perichondrium, the hyaline cartilage was collected.The MSCs and elastic chondrocytes, which were acquired by purification, were cultured in vitro passage.After purifying and culturing MSCs and elastic chondrocytes to the 3rd generation, the general RNA of MSCs, elastic chondrocytes, and hyaline cartilages were taken by the One-step Method and inversely transcribed into double-chain CDNA. With separately marking by Cy5-dCTP and Cy3-dCTP, the cDNA probes of MSCs and chondrocytes were crossed with two chips and strictly cleaned. After scanning by the double channel laser scanner LuxScan 10KA, the fluorescent signal images were analyzed by the graphical analysis computer software GenePix Pro4.0.Using the Twice Significance of Difference as the standard, that was, cy5/cy3<0.5 meant the up-regulated gene, cy5/cy3>2 meant the down-regulated gene. The differential representing genes were found. Considering the gene function and the ratio of cy5 to cy3, then the key genes in inducing the differentiation from MSCs to chondrocytes were found.Results:After testing the acquired MSCs , the results met the requirement of the experiment.The OD260/OD280 of the general RNA of the MSCs and two kinds of chondrocytes were all between 1.80 and 2.0. Moreover, the electrophoretic bands of the three groups of RNA were clear. The brightness between 28S and 18S was close to 2 to1. The purity of the three samples met the requirement and without degradation.Among the 2148 differential representing genes which were selected from the hyaline chondrocytes group, there were 1041 up-regulated genes and 1107 down-regulated genes.Among the 2114 differential representing genes which were selected from the elastic chondrocytes group, there were 1226 up-regulated genes and 888 down-regulated genes. Totally there were 582 same up-regulated genes in these two groups, and 612 same down-regulated ones. While the number of genes which were up-regulated in the hyaline chondrocytes group and at the same time, down-regulated in the elastic chondrocytes group were 459, and the number of genes which were up-regulated in the elastic chondrocytes group and at the same time, down-regulated in the hyaline chondrocytes group were 345.Considering the gene function and the ratio of cy5 to cy3, Chm1(chondromodulin1), CTGF (connective tissue growth factor) ,Col2α1(procollagen, type2, alpha1), Cart1(Cartilage homeoprotein 1), MMP3(matrix metallopeptidase3) as well as TIMP3 (tissue in hibitors of metalloproteinase3, TIMP3) were designated as the target genes. Conclusion:There were significant differences in the gene expression profile of the MSCs and chondrocytes.The induction of differentiation from MSCs to chondrocytes could be realized by up-regulating or down-regulating the representations of centain key genes though the cooperation of different kinds of growth factors.Chm1 and CTGF, the up-regulated genes compared with MSCs in both hyaline cartilage and elastic chondrocytes, might be the key factors in inducing the diff- erentiation from MSCs to chondrocytes.Col2α1 and Cart1 which up-regulate compared with MSCs in hyaline chondrocytes group and down-regulate compared with MSCs in elastic chondrocytes group, might be the key factors in inducing the differentiation from MSCs to hyaline chondrocytes.MMP3 and TIMP3, which up-regulate compared with MSCs in elastic chondrocytes group and down-regulate compared with MSCs in hyaline chondrocytes group, might be the key factors in inducing the differentiation from MSCs to elastic chondrocytes.After further analyzing and testing the results of the acquired differential representing genes mentioned above, the new growth factors might be selected, and the oriented induction from MSCs to the well shaped and stable tissue engineering cartilage might be realized.