The Study Of The Conditions And Molecular Mechanism Of The Differentiation From The Synovial Mesenchymal Stem Cells To The Fibrocartilage

PURPOSE Explore the necessary and sufficient conditions for the SMSCs’ differentiation to the fibrochondrocytes, and repeat this process in vitro. Then explore the molecular mechanism of this process. METHOD Obtain and purify the SMSCs with the attachment culture and the limiting dilution method. Identification of the stem cells use the morphological method, ultrastructure, ultrastructure, cell dynamics process and multipotential differentiation. And the stability, the security, the chondrogenic potential and the cytoactive were focused on. After the preliminary experiment, the review of literature and the imitation of the internal environment of the knee a L60(212) orthogonal experiment were used to seek for the necessary and sufficient conditions. The dependent variable of the orthogonal experiment was the transformation rate of the fibrochondrocytes. The specific gene expression of the differentiation and the chondrocyte were detected using the quantitative RT-PCR, and the special molecule of the cells were test with the specific staining such as the toluidine blue, and then the rate were count on the cells. The steady and fittest fibrochondrogenisis process were confirmed at last after trial and error, and the gene expression of the process were explored. Some gene microarrays with8697genes were built according to the literature review,2153of them come from the expressed sequence-tag (EST) library and others come from the Genebank of NCBI database. The open reading frame of some genes is too short to use in the microarrays and there are8605genes were test at last. The differential expression genes were checked with the RT-PCR. The quality control were operate with the Quackenbush, and data analyze using the method described by the Eisen. The lower limit of the differential expression is more than2fold. The function analyze of the gene were proceed. Finally we evolve that the TGFβ-TGFPreceptor-Smad2/3pathway and the BMP-BMP receptor-Smad1/5/8pathway may participate in the SMSCs’fibrochondrogenisis according to the gene expression and the regulation environments. The RT-PCR, Western-Blotting and the specific stain method were used to explore the relationship between the regulatory factor and the fibrochondrogenic genes. RESULT The SMSCs were steady, safety, activity, easy to obtain and more potential for the fibrochondrogensis. The key factor of the fibrochondrogenic process include the TGF(31,BMP7, ASA, bFGF, and the IGF. The time related accommodation of these factor can regulate the process of the SMSCs’fibrochondrogensis, the other factors are interactive with these factors and less remarkable. Others environments such as mechanical environment and oxygen concentration were significant modulations. Microarrays demonstrate that there are1361genes differential expression in the process, and the abundance show time related changing. Clustering analysis show the21st day of the cells is similar to the SMSCs, and the gene expression of the3ed、7th、14th day is similar. The function analyze show the proliferation-associated gene of primary cells and the differentiated cells are high expression, but the function of most of the genes is uncertain. The TGFβ-TGFβreceptor-Smad2/3pathway and the BMP-BMP receptor-Smad1/5/8pathway are two important regulatory pathway, besides the glucose level and the mechanical environment modulate the process. More regulatory pathway and more unknown factors needs to be explore. CONCLUSION SMSCs is the appropriate seed cells for meniscus tissue engineering. Rational environment can get the experimental model for the SMSCs’fibrochondrogensis. A great amount of genes differential expression in the process. Glucose level and the mechanical environment modulate the fibrochondrogensis process with the TGFβ-TGFβreceptor-Smad2/3pathway and the BMP-BMP receptor-Smad1/5/8pathway. There are interaction between these pathways, the relations between the regulatory pathway and the gene expression is quite complex. The down-stream gene of the pathway is regulated by these modulations, the gene expression also regulate the messagers and the receptors. At the same time, some miRNA regulate the genes abundance. The molecular mechanism is still unclear, more studies should be carried out.