| ObjectiveCartilage injury is one of the most common and serious diseases and a major cause of disability to some extent, which can also causing the social and family cost burden. The previous methods to repair the articular cartilage defects still do not attain satisfying results because of the limited self-repair ability after injury, and reconstruction of cartilage defects remains one of the most difficult clinic problems for joint surgeons. Recently,the sharp development of gene-engineering and tissue engineering provides great chances to deal with this difficult challenge. The goal of our study is to construct the lentiviral expression vector of TGF-β1and IL-10gene and transfect them to BMSCs, differentiate bone marrow-derived mesenchymal stem cells into chondrogenesis, to provide the theory basis for the differentiation of stem cell to specialized cell types through the induction of multiple genes and experimental data for constructing tissue engineering cartilage.Materials and Methods1Healthy New Zealand white rabbits, approximately2-3months of age were used in this study. A6-8ml of bone marrow was isolated from the both Iliac and tibial shaft of each rabbit. The bone marrow-derived mesenchymal stem cells (BMSCs) suspension was aspired after bone marrow was centrifuged by percoll separating medium.They were primarily cultured and subcultured in vitro, and then divided into four groups according to the difference of lentivirus vectors:group A receiving transforming growth factor β1(TGF-β1); group B receiving TGF-β1and Interleukin-10(IL-10); group C empty vector transfection; group D receiving no cell growth factor.2Construct the lentiviral expression vector of TGF-β1and IL-10gene and transfect them to BMSCs, then make sure the MOI (multiplicity of infection) and infection time needed.3According to the transfect situation and addition of the cytokine, the BMSCs were divided into6groups:group A receiving transforming growth factor β1(TGF-β1); group B receiving TGF-β1and Interleukin-1β(IL-1β);group C receiving TGF-β1and Interleukin-10(IL-10); group D receiving TGF-β1+IL-10+IL-1β;group E empty vector transfection; group F receiving no transfection. GroupsA-E were cultured conventionally ina-MEM medium containing10-7mmol/ml Dex,50ug/ml Vitc, and10%fetal bovine serum. BMSCs were collected from the6groups,which mRNA were extracted to detect the expression of SOX-9,Aggrecan and Type Ⅱ collagen by real time PCR and which protein were extracted to detect the expression of Type Ⅱ collagen.Results1.Changes of cell morphology The growth of the BMSCs was observed with the phase contrast microscope. During the beginning of24hours, sparse primary cells fastened wall, the shape of the primary BMSCs was short spear-like. Most cells had fastened wall and began to extent and split up, and clusters which formed of single cell division appeared after48hours, most of the floating cells were cleared after changing medium of1-2times.5-7days later, the cell counting showed up along with division and proliferation of cells. The subcultured cells grew faster. After about5days, monolayer cells with high nuclears and many grains in cytoplasm, were observed in the shape of parallel or swirl. 2Transfect the lentiviral expression vector of TGF-β1and IL-10gene to BMSCs, and fluorescence expression was found after12h, and the transfection efficiency was about70%in the condition of MOI=100after96h.3Real time PCR and western blotting detection SOX-9、aggrecan and Type Ⅱ collagen in group A-E all expressed in the day7and14. The levels of three genes expression in group A、C were higher than that in opposite group adding IL-1β (group B and D) and group E,which shew statistical difference. The levels of three genes expression in group B were higher than that in group D, which shew statistical difference. While the level of three genes expression in group A and C had no statistical difference.Conclusion1BMSCs are the ideal seed cells for constructing tissue engineered cartilage.BMSCs can be easily obtained, separated and generated.their characteristics are stable in vitro. BMSCs can be induced to cartilage cells under some conditions in vitro2expression of target gene in BMSCs by lentiviral vector was high efficient and stable.3Cytokines play an important role in cell proliferation and chondrogenic differentiation.have synergy effect in the differentiation from BMSCs into chondrocytes. TGF-(31have synergy effect in the differentiation from BMSCs into chondrocytes. IL-10with cartilage protection function may help against IL-1β with inhibition of cartilage synthesis. |
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