To Construct Recombinant Lentivirus Vectors Of BMP-2Gene And Study About BMSCs Osteochondral Differentiation After Transfected

1.To construct recombinant lentivirus vectors of BMP-2gene.2.LV-BMP-2and LV-TGF-β1gene transfect and induce BMSCs differentiation intoosteoblasts and chondroblasts,provide new thought to explore the bone tissue engine-ering in the construction of the cartilage.1.Construct recombinant lentivirus vectors of BMP-2gene:The BMP-2cDNA wereextracted and packed into lentivirus vector and transfected the293T cells throughliposome Lipofectamine200.Porcine BMP-2gene in the vector PcDNA3.BMP-2was identified enzyme digestion and DNA sequencing,and the lentiviral titer was detected by Real-time PCR;The express activity of BMP-2gene in293T cells were checked by western blot.2.BMSCs were transfected by recombinant lentivirus vectors of BMP-2gene,then the multiplicities of infection(MOI) was determined by expressions of fluorescence andthe expressions of BMP-2in the infected BMSCs were detected by immunohistoche-misty,Real-time PCR,Western blot and ELISA.MTT test was used to assay the proliferation of transfected BMSCs; ALP staining,alp activity detection and lipo-form-ation were used to analysis the BMSCs ossification ability;recombinant lentivirus vectors of TGF-β1gene transfect the BMSCs,for a times foster,then the capacity of cartilage differentiation was detected by Collagen type Ⅱ immunohistoch-emisty and RT-PCR.1.The BMP-2recombinant lentiviral vectors was constructed successfully.It was absolutely correct by enzyme digestion and sequencing identification,and it could transfect293T cells and expression.The lentiviral titer was4.07×108TU/m.2.The BMP-2recombinant lentiviral vector transfect BMSCs efficient;the best MOI that BMP-2recombined lentivirus infected BMSCs was100,and the expressions of BMP-2mRNA and protein in infected BMSCs were stable and high,immunohistochemisty and RT-PCR show the BMSC express Collagen type Ⅱ mRNA and protein.The BMP-2recombinant lentiviral vector was constructed successfully,the expressio-ns of BMP-2mRNA and protein in infected BMSCs were stable and high,the BMSCs can be induced into osteoblasts and chondroblasts after transfected by BMP-2and TGF-β1.