The Differentially Expressed Genes Between Rat Mesenchymal Stem Cells And Elastic Chondrocyte

Objective: To screen differentially expressed genes between rat mesenchymal stem cells and elastic chondrocyte by microarray and search the biotic factors which can induce chondrocyte differentiation of MSCs in order to get tissue engineered cartilage.Methods: MSCs were got through the combined utilization of density gradient centrifugation and adherence screening methods. The chondrocytes were got from rat ear cartilage by enzyme digestion. MSCs and chondrocytes were cultured with DMEM and DMEM-F12 medium of foetus of serum with the volume fraction of 0.1 in vitro. Cell RNA was obtained with total RNA extraction from the cells of 2nd- 3rd generation. Both of RNAs were reversely transcribed to cDNAs and labeled with the fluorescent Cy5-dCTP and Cy3-dCTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After high stringent washing ,the fluorescent signals were canned by LuxScan 10KA scanner. The values of Cy5-dCTP and Cy3-dCTP each spot were analyzed and calculated by GenePix Pro 4.0 software.Results: The absorbance of the samples of MSCs and cartilage A260/ A280 is more than 1.79.The total quality of chondrocyte and MSCs are 79.2μg and 22.5μg.The electrophoresis bands of two kinds of RNAs are distinct. The ratio of 28S and 18s RNAs bands brightness is close to 2:1.By applying this cDNA microarray, 2114 differentially expressed genes in chondrocyte, whose ratios of Cy5/Cy3 were higher than 2.0 or lower than 0.5, were screened out among the 28800 target genes. Among the differentially expressed genes, up regulated and down regulated genes were 1226 and 888 respectively. The remarkably differentially expressed geneswhose ratio are more than 20 were mainly analysed. It were defined as remarkably differentially expressed genes including cell skeleton and cinetc-protein related genes, cell cycle related genes, immune related genes, signal transduction related genes, extracellular matrix related genes, transcription factors related genes, protein translation and synthesis related genes, metabolism related genes. Two growth factors which have never reported were found over expressed in cartilage, named chondromodulin and connective tissue growth factor. The characteristic genes of cartilage in extracellular matrix up regulated express in cartilage, for example, collagen, type II, elastin, while the genes over expressing in MSCs are mostly enzymes, proved to have the ability of differentiating into various tissues.Conclusion:(1)There is remarkable difference between gene expression profiles of rat mesenchymal stem cells and elastic chondrocyte.( 2 ) It is probable that kinds of biotic factors take effect on chondrocyte differentiation of MSCs, up regulating and down regulating some key genes.(3)Twelve hundred and twenty six genes were found over expressing in cartilage, chondromodulin and connective tissue growth factor as delegates, which are possibly related to chondrocyte differentiation of MSCs.(4)Eight hundred and eighty eight genes were found down expressing in MSCs, MMP9 as delegate. It is important to inhibit the effect of that in chondrocyte differentiation of MSCs.