The Research of7,8-dihydroxy Coumarin Promotes Proliferation and Chondrogenic Differentiation of Rat Adipose-derived Mesenchymal Stem Cells
|Keywords||Adipose-derived mesenchymal stem cell chondrogenic differentiation proliferation 7,8-dihydroxy coumarin|
In the plastic surgery, often meet for cartilage transplantation, trauma ofcartilage damage and defect. The articular cartilage has no nourishment fromblood vessels and surrounding tissues, in addition to the limited proliferationability of chondrocytes not to be transplanted, so it is hard to repair the damageof the articular cartilage. Currently, the clinical treatments of cartilage damageinclude arthroscopic lavage debridement, subchondral bone drilling,microfractures technology, periosteum or perichondrium transplantation, andautologous or allogeneic bone and cartilage transplantation. However, thesemethods have the poor prognosis, not restore the articular cartilage into thenormal tissue and biomechanical characteristics in the true sense. The tissueengineering has applied the biological substitutes to repair the damaged tissues.The autologous chondrocyte is the only clinical cartilage seed cell applied in thetissue engineering. However, the donor source is limited, the cells’ isolation andculture are difficult, the cells are of poor proliferation capacity, and thus theapplication is limited. As to the embryonic stem cells and the inducedpluripotent stem cells, because of the ethical issues and safety issues, there isstill a long-range from the clinical application. Adult stem cells are widelypresent in the body, especially adipose-derived mesenchymal stem cells(ADMSCs), with a wide distribution to obtain easily, are ideal for the use as thetissue engineered seed cells.For tissue engineering cartilage prerequisite is how to choose and obtainseed cells, this to how to construct the structure of organizations play a decisive role. At present, clinical applications of tissue engineering cartilage seed cellonly autologous cartilage cells. But there are many defects, for area is limited,separation cultivate difficulties, the in vitro culture, proliferation ability islimited, etc. At the same time because pluripotent stem cells and embryonicstem cells induced in the application of safety, ethics, etc and clinical applicationhad a certain gap. And autologous stem cell especially fat source ofmesenchymal stem cells in the body are widely distributed, its source is more,acquisition methods relative simple, as tissue engineering seed cells of the idealchoice.However, how to make the stem cells in vitro quickly and a large numberof breeder? At the same time, how to make it more effective to the cartilage celldifferentiation?Now routine use of laboratory to promote stem cells value-added revulsantis all sorts of cell factor, and the cell factor extraction and separation make itscost is expensive, and human internal environment is difficult to rely on cellfactor to simulation. With the deep research of natural drugs, which facilitatedthe role of bone also concern?Previous studies have shown that7,8-dihydroxy coumarin, a natural drugcan promote bone formation and increase bone mineral density. Tang et al.(2008) reported that coumarin and its derivatives can activate the p38-andERK-dependent pathways in rat osteoblasts, raise the BMP-2expression andpromote bone formation. Tang et al (2010) reported that coumarin compoundsosthole is able to activate the Wnt/beta-catenin-BMP2signaling pathways inmouse osteoblasts, promote osteoblast and contribute substantially to the mouse skull bone repair and bone mineral densityTherefore, this study used7,8-dihydroxy Coumarin (hereinafter referredto as Coumarin, Coumarin) training rats adipose mesenchymal stem cells(ADMSCs), so as to research in Coumarin in promoting fat stem cells into therole of cartilage differentiation.This experiment adopted type I collagen enzyme digestion, adherent culturemethod rats to fat tissue separation culture ADMSCs and carry on thepurification. Experiments show this method can in rats subcutaneous adiposetissue sources of sample separation and induced cultivate long spindle andattached growth adipose mesenchymal stem cells. Cultivate cellsIntensive growth, a fish sample, multiplication rate faster, doubling time is46.7h. After flow cell technical detection and the results showed that thecultivated cells can express CD73, CD29, CD90and CD105antigen, and CD14,CD34, CD45, CD79alpha and HLA-DR did not see expression. In the appraisalof mesenchymal stem cells function, the multiplex differentiation potential.thisis one of the important indexes, this study applies different kinds of revulsivewill mesenchymal stem cells to three kinds of cell differentiation, respectively:osteoblast, cartilage cells and fat cells to prove its changeover differentiationpotential, the results showed that conform to the mesenchymal stem cellsappraisal standard.After complete the above appraisal, we use the cell micelle training method,observation coumarin to ADMSCs to cartilage differentiation role. The resultsof the study show that used only coumarin, induced into cartilage differentiationis micro, and coumarin and TGF-β1has synergistic effect, the intracellular SOX9、PGC-1α、Col2a1three kinds of gene expression has more obvious role,can promote the extracellular matrix type ii collagen and glycosaminoglycangathering. Mesenchymal stem cells to fat to the cartilage cell differentiation hasrole in promoting. And this study also found that an interesting phenomenon thatalong with the concentration change, has the function of cartilage differentiationability to change, through test confirmed the most appropriate concentration is50ug/ml-100ug/ml. Speculation its possible mechanism for: coumarin in richnatural cell factor and oligopeptides component can promote cell or cells andextracellular matrix interaction to promote ADMSCs to cartilage celldifferentiation.