| Background The damage and degeneration of cartilage caused by tumors, trauma, inflammation, metabolic abnormalities is one of the most common and challenging topics in orthopaedy management. New advance in cartilage tissue engineering for cartilage repair brings a ray of hope to people, becomes a research focus. As seed cells, bone marrow mesenchymal stem cells (BMSCs) with multilineage differentiation potential and strong self-renewal ability have being studied extensively. But some problems of such as not fully understanding repair mechanism, the improvement of induction methods of cartilage differentiation hindered its implication.microRNA (miRNA) is a group of endogenous non-coding single-stranded small RNA species 22 nucleotides in length, which take part in posttranscriptional regulation of gene expression. In recent years, studies have shown that miRNAs have been shown to be involved in regulating generally individual development, proliferation, differentiation and other biological processes. Especially its influence in self-renewal and differentiation,maturation and maintenance of biological characteristics of stem cells including BMSCs has attracted increasing attention. We had screened differentially expressed miRNA genes between human BMSCs and chondrocytes by microarray in preliminary studies in our laboratory .Objective To detect and validate the miRNAs which specifically expresses in BMSCs-derived chondrocytes differentiation and to study their biological functions for demonstrating further how they play a role in regulating differentiation process.Medthods (1) The human BMSCs were obtained in bone marrow aspirated from adult were isolated by the whole marrow-adherence way from the mononuclear cells,and were cultured in vitro.Then the morphology,phenotype, cell cycle was investigated for the knowledge of the characteristics of BMSCs by using a flow cytometer and inverted microscopy for identification. BMSCs were separately induced into adipocytes and osteocytes in vitro so as to prove the multipotent ability of BMSCs by oil red staining and ALP staining and alizarin red S stainingcalcium staining. (2) The BMSCs then were induced into chondrocyte in monolayer by a chemically defined medium including1ng/ml TGF-β1, 0.2mM ascorbic acidy,6.25ug/ml transferrin, 1.25ug/mL BSA,10-7M dexamethasoney, 6.25ug/ml insulin. BMSCs were cultured in monolayer in the conventional culture medium at the same time as negative control. After 21-day's chondrogenic inducement, the cells were analysed by morphology observation, staining with haematoxylin-eosin and toluidine blue, immunohistochemistry evaluation with the monoclonal antibody specific to type II collagen. (3) The miRNAs which differentiated expressed miRNAs between BMSCs and BMSCs-derived chondrocytes possibly play an important role in differentiation process with computational softeware online. Four interested miRNAs were detected by read time RT-PCR ,they were hsa-miR-26b, hsa-miR-28,hsa-miR-130b,hsa-miR-152. (4)The function of miRNA-26b on chondrocytes differentiation was investigated by transfecting BMSCs with one's mimics and inhibitor and analyseing the cells which were cultured for 21 days by staining with haematoxylin-eosin and toluidine blue, immunohistochemistry evaluation with the monoclonal antibody specific to type II collagen.Results (1) BMSCs which were isolated from bone marrow was detected by flow cytometer, phenotypes showed CD29, CD44surface antigens of culture cells was positive, but CD34, CD45surface antigens was negative, that and morphological characteristics ,result of cell cycle analyse, differentiation into adipocytes and osteocytes all basic features indicated these cells were human BMSCs. (2) BMSCs were induced in monolayer condition for 21 days by defined medium were metachromasia by toluidine blue and could be identified by positive staining for type II collagen. (3) Based on miRNA expression miRNA profiles by miRNA microarray technology , we verify four miRNAs that were significantly up-regulated expression of the human BMSCs and BMSCs-derived chondrocytes by information biology and using real time RT-PCR assay. (4) BMSCs were observed with positive results of HE staining, toluidine blue, type II collagen immunohistochemistry evaluation after transfection of miR-26b mimics, while were not blocked into cartilage after transfection of miR-26b inhibitor which were cultured with chondrogenic Supplement.Conclusion (1) BMSCs which can be obtained from bone marrow by using the whole marrow-adherence way and cultured in vitro can be induced into the chondrocytes. (2) There are several miRNAs significantly differential expressed between BMSCs and BMSCs-derived chondrocytes which may play a role on regulating the chondrogenic pocedure of BMSCs,but this regulation can be effected even can be inhibited by internal and external environmental factors. (3) The miR-26b can promote the differentiation of BMSCs into chondrocytes without chondrogenic Supplement.
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