The Vitro Study Of Injectable Engineered TGF-beta3 Recombinant Chondrocytes In The Treatment Of OA

Objective: Exploration of bone marrow mesenchymal stem cells develop into cartilage cell differentiation. construction of injectable engineering TGF-beta3 plasmids, the cartilage cells were transfected to build sustainable secretion expression of TGF-beta3 protein, for later target to repair rat osteoarthritis cartilage injury research provides experimental basis. Methods: 1. Derived from rat bone marrow mesenchymal stem cells(BMSCs), DMEM/F12 culture medium was used to culture, and the subculture was further separated and purified; In P3 by anti-CD29, anti-CD45 and anti-CD90 were identified BMSCs surface antigens, using CCK-8 assay P3 generation of cell proliferation activity. The P3 generation of third day were randomly divided into two groups: the induction group and the control group. In the induction group, the culture medium was used as the chondrocyte culture medium, and the control group was treated with 10% serum DMEM/F12 culture solution. Respectively in the 4d, 7d, 14 d by immune fluorescence cytochemistry and alcian blue staining were used to detect the expression of each group of type II collagen(Col II) and aggrecan.2. The artificial synthesis of matrix metalloproteinases(MMP) complementary nucleotide chain was inserted containing enhanced green fluorescent protein(EGFP) pcDNA3.1(+) plasmid, recombinant plasmid pcDNA3.1(+)-EGFP-MMP double enzyme digestion was assayed with gel electrophoresis; With Reverse Transcription-Polymerase Chain Reaction(RT-PCR), extracting respectively Latent Associated Protein(LAP) and the activity of TGF-beta3(mTGF-beta3) of rat. Target genes LAP and mTGF-beta3 were inserted into the upstream and downstream of MMP in sequence, recombinant plasmid pcDNA3.1(+)-EGFP-LAP-MMP and recombinant plasmid pcDNA3.1(+)-EGFP-LAPMMP-mTGF-beta3 in turn double enzyme digestion were assayed for gel electrophoresis; Recombinant plasmid pcDNA3.1(+)-EGFP-LAP-MMP-mTGF-beta3 was detected for Gene sequencing.3. The induced cells were randomly divided into three groups: empty plasmid group, recombinant plasmid group and blank control group. By liposome transfection method, the recombinant plasmid pcDNA3.1(+)-EGFP-LAP-MMP-mTGF-beta3 was transfected into cells, the inverted fluorescence microscope observated its expression in the cell; Through Western blot analysising and comparing the TGF-beta3 protein expression within cells of the recombinant plasmid transfected group and empty plasmid transfected group. Extracting cell culture solution of the recombinant plasmid transfected group, randomly divided into MMP group and non MMP group, MMP group was added into MMP2, and no MMP group was added with equal amount of PBS, 2 hour later, Western Blot was analysised expression of mTGF-beta3 protein. Results: 1. Acquired large amounts of purified BMSCs. BMSCs in P3 generation of the third day has maximum logarithm proliferation activity; From immune fluorescence cytochemistry and alcian blue staining showed that BMSCs can differentiate into cartilage cells, successfully access to the cartilage cells.2. Gel electrophoresis results of RT-PCR are shown to acquire the LAP and mTGF-beta3 consistent with reported size; Geting into the recombinant plasmid pcDNA3.1(+)-EGFP-LAP-MMP-mTGF-beta3 of complete inserting the engineering of TGF-beta3 gene sequence.Double restriction enzyme digestion bands of plasmid pcDNA3.1(+)-EGFP-MMP was consistent as size of MMP. The recombinant plasmid pcDNA3.1(+)-EGFP-LAP-MMP and the recombinant plasmid pcDNA3.1(+)-EGFP-LAPMMP-mTGF-beta3 respectively double enzyme cutting bands were the same as size of LAP and mTGF-beta3; Gene sequencing result accords with GenBank gene sequence with rat LAP and mTGF-beta3.3. Under inverted fluorescence microscope found that the recombinant plasmid group transfected by pcDNA3.1(+)-EGFP-LAP-MMP-mTGF-beta3 could express green fluorescent protein in cells; Western blot result showed the recombinant plasmid transfected cells comparing with empty plasmid transfection group has better significant expression of TGF-beta3 protein, Comparison of the two groups was statistically significant(P<0.05); Western blot result showed mTGF-beta3 protein expression of the MMP group is higher than that without the MMP group. Conclusion: 1. BMSCs in vitro could be rapidly induced into chondrocytes.2. The recombinant eukaryotic expression plasmid pcDNA3.1(+)-EGFP-LAP-MMPmTGF-beta3 was successfully constructed.3. Recombinant plasmid pcDNA3.1(+)-EGFP-LAP-MMP-mTGF-beta3 was transfect- ed into chondrocytes to express high expression of mTGF-beta3 protein.