| Objective This study was to establish the experimental method for isolating, culturing, proliferating and inducing rat bone marrow mesenchymal stem cells (BMSCs) in vitro, to construct portable FGF4 signal peptide for promoting secretion of basic fibroblast growth factor (BFGF) and to enhance green fluorescent protein (EGFP) of the lentiviral vector (LVs); to observe FGF4-BFGF & EGFP fusion gene recombinant lentiviral vector-modified BMSCs for repairing myocardial infarction (MI) and its related mechanism.Methods The method for isolating, cultivating, proliferating and inducing BMSCs: Bone marrow was taken out from the bilateral femora and tibiae, and cultured with L-DMEM culture solution in culture bottles. MSCs were isolated and purified by the wall-adhering method. The morphology and graph of every generation were observed under the phase-contrast microscope. And the third-generation (P3) cells were collected to assay by the flow cytometry technical appraisal. The pGC-FU lentiviral vector that carries the EGFP was chosen as a medium of gene transduction system. In-Fusion technology was adopted to construct the promoted secretion of FGF4 signal peptide and BFGF gene recombinant lentiviral vector, which was identified by the polymerase chain reaction (PCR), enzyme digestion and sequencing.. The 293T cells were transfected through the liposome Lipofectamine 2000. The lentiviral titer was detected by real-time fluorescence quantitative PCR (RT Q-PCR). The optimum infection plural (MOI) and the secretion of BFGF protein expression level were determined by expression of fluorescence and enzyme linked immunosorbent assay (ELISA). The rat model of myocardial infarction was set up by the coronary artery ligation . The 120 rats were randomly divided into sham operation group (Sham group), solvent control group (Vehic group), no-load lentiviral group (EGFP / BMSCs group), BFGF fusion gene lentivirus group (BFGF / BMSCs) and FGF4 - BFGF4 fusion gene lentivirus group (FGF4-BFGF/BMSCs group). The 24 rats of each group were divided into four sub-groups on the 3rd d, 7th d, 14th d, and 28th d after the operation. The local myocardial infarct area of the corresponding experimental animals was injected with 100ul 1×PBS solution or 2×106 cells after the model of myocardial infarction was set up for 30 - 60min. The Yihong - hematoxylin (Hematoxylin Eosin, HE) staining was used to observe the myocardial pathological changes after transplantation, and the laser scanning confocal microscope (LSCM) was used to observe the transplanted cell survival and differentiation. Western-blot was adopted to detect the infracted myocardial tissue BFGF protein expression of the experimental rats, and immunohistochemistry (IHC) was adopted to detect the positive expression of Factor VIII. Western-blot and Real Time Q-PCR were adopted to detect the apoptosis regulatory genes Bcl - 2, Bax mRNA and protein expression. In situ nick end labeling (TdT-mediated dUTP nick end labeling, TUNEL) was adopted to detect apoptotic index Detect Change. IHC was adopted to detect the protein expression of MMP-9, TIMP-1. Masson staining was adopted to detect the myocardial collagen fiber changes, and parallel digital high frame rate echocardiography was adopted to observe the conditions of cardiac function of the experimental rats after 28 days of the cell transplantation.Results Primary cultured BMSCs adhered to and stretched into polygon or spindle within 24 hours. The first 3 days was the relative inhibition period, and then the cell proliferation accelerated gradually in a pattern of logarithm and reached the period of plateau by Day 7 or so, with a 90% cell coverage. The number of P3 BMSCs can reach 106 2 weeks later, and that can satisfy the required number of cell transplantation. The signs of BMSCs with positive CD29, CD44, CD90 and negative CD34, CD45 were detected by the flow cytometry, and that is consisted with the characteristics of BMSCs. In-Fusion technology was adopted to construct the FGF4-BFGF fusion gene recombinant lentiviral vector. The lentiviral vector was identified entirely correct by PCR, enzyme digestion and sequencing, and the transfected 293T cells were identified to express correctly. The optimum MOI of BMSCs was 10 with the 1×10~8TU/ml lentiviral titer. BMSCs infected by FGF4-BFGF fusion gene recombinant lentiviral vector can express and secrete BFGF protein efficiently. Compared with Sham group, the myocardial tissue pathological changes were significantly lighter in the FGF4-BFGF/BMSCs group than in other groups, on the 3rd d, 7th d, 14th d, 28th d after the myocardial infarction model was set up. The survival number of transplanted cells was significantly larger in this group (P <0.05) than in other groups on the 28th d of the coronary ligation, but there were few cardiomyocyte-like cells visible except some transplanted cells with the host myocardial cell fusion. In addition, BFGF, Bcl-2, TIMP-1 were up-regulated, whereas Bax MMP-9 levels were decreased; the microvessel density was noticeably increased, whereas the apoptotic index was significantly reduced in the myocardial infarct areas of Vehic group, EGFP / BMSCs group, BFGF / BMSCs group, and FGF4-BFGF/BMSCs Group. These changes were of statistical significance (P <0.05). Compared with the other groups, collagen fibers in the myocardial infarct area decreased, myocardial cells increased and the left ventricular wall was thickened in FGF4-BFGF/BMSCs group. The cardiac function was significantly improved on the 28th d after transplantation (P <0.05), close to that of the shame group.Conclusion Highly purified BMSCs can be separated by adherent Screening method. BMSCs proliferate quickly in vitro. Due to their differentiations, BMSCs are good seed cells for treating myocardial infarction through transplantation. In-Fusion technology can be adopted to construct the FGF4-BFGF & EGFP fusion gene recombinant lentiviral vector, which can express and secrete BFGF protein efficiently. The modified BMSCs improve the rat cardiac function after myocardial infarction via angiogenesis promotion, anti-cardiomyocyte apoptosis and ventricular remodeling inhibition.
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