| Objective Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has been proposed as a potential therapeutic strategy for post-infarction left ventricular dysfunction. However, marginal recovery of cardiac function has been resulted from considerable cells death after transplantation into the infarct myocardium. Here, we genetically modified BMSCs with anti-apoptotic and pro-angiogenesis Survivin (SVV) gene and evaluated cell survival, angiogenesis, collagen deposition and functional improvement in a rat model of myocardial infarction.Methods 1. BMSCs were prepared from the bone marrow of rats based on their adherence to tissue culture surface. The growth properties were compared by cell counting between successively passaged BMSCs (P1, P2, P3). P3 BMSCs were further identified by fluorescence-activated cell sorter (FACS) for CD14, CD29, CD34, CD44, CD45, CD90 antibodies. 2. p-FUGW, a lentiviral vector with green fluorescent protein (GFP) used as a tool of gene delivery, SVV recombinant lentiviral vector (p-FUGW-SVV) was constructed by infusion technology, and then identified by polymerase chain reaction (PCR), enzyme cutting and sequencing. After p-FUGW or p-FUGW-SVV was cotransfected with another two helper plasmids (p-Helper1.0 and p-Helper2.0) into 293T cells respectively, virus supernatant was collected and condensed, and then the titer was assayed by multi-proportion dilution. P1 BMSCS were infected by SVV recombinant virus at MOIs of 2, 8 and 32, respectively. BMSCs infected by Mock-virus (p-FUGW) were used as control. Protein expression of SVV was detected by Western-blot and compared between P1 BMSCs infected at different MOIs. 3. After infected by SVV recombinant virus or Mock-virus, P1 BMSCs were passaged to P3, which was named as SVV/GFP+BMSCs or GFP+BMSCs, respectively, and then, the GFP-positive cells were quantified by FACS. After they were cultured for 0 hour, 12, 24, 48 and 72 hours in serum-deprivation medium, the apoptotic cells were quantified by FACS. Uninfected BMSCs were used as control. 4. In vivo, SVV/GFP+BMSCs were injected after infarction. Rats treated with PBS were used as control (vehic group). The surviving cells were tracked and their differentiation into cardiomyocyte-like cells evaluated by immunofluorescent staining after transplantation. Expression of bFGF, HGF, VEGF in the infarct zone were assay by reverse transcript (RT)-PCR, immunostaining or western-blot, and pathological changes were detected by HE staining at 7, 14, 28 days after BMSCs transplanted. Angiogenesis was detected by immunnostaning for vWF antibody, collagen deposition detected by Masson staining and quantified by second harmonic generation imaging at 28 days after BMSCs transplantation. Recovery of cardiac function was assessed by ultrasoundcardiogram at 7, 14, 28 days after BMSCs transplanted, and the cardiac function at 1 day after transplantation was used as baseline.Results 1. A gradual loss of replication ability was companied with BMSCs passaged from P1 to P3. P3 BMSCs were CD29, CD90, CD44-positive and CD14, CD34, CD45-negtive. The sequence of SVV which contained in FUGW-SVV plasmid was correct. 293T cells emitted green fluorescence under phase-contrast fluorescent microscope after they were transfected with p-FUGW-SVV. 2. The virus titer of the packaged lentivirus was about 1.5×108TU/ml. A gradual enhancement of fluorescent intensity of the infected P1 BMSCs was companied with MOIs increased. More than 95% of successively passaged P3 BMSCs were GFP-positive. Two protein styles, i.e., SVV/GFP fusion protein and a morphon of SVV protein, were observed, and expression of protein morphon was highest when BMSCs were infected at MOIs of 8. 3. The number of apoptotic cells was time-dependently increased. But the cell apoptosis in SVV/GFP+BMSCs group was significantly lower than in GFP+BMSCs group (P﹤0.05). Especially, SVV overexpression reduced BMSCs apoptosis by about 25% after cultured for 72 hours in serum-deprivation medium when compared with GFP+BMSCs group. There were not differences at any time points between GFP+BMSCs and the uninfected BMSCs (P﹥0.05). 4. Transplantation with SVV/GFP+BMSCs increased about 3-fold, 6-fold of the cellular survival at 7 days and 28 days respectively when compared with GFP+BMSCs group (P﹤0.05). Only few transplanted BMSCs fusion with host cardiomyocytes but not differrention were observed. Expression of bFGF, HGF, VEGF in the infarct zone was higher in GFP+BMSCs group only at 7 days after transplantation than in vehic group. Expression of these growth factors in SVV/GFP+BMSCs was not only higher than vehic group but also than in GFP+BMSCs group(P﹤0.05), especially at 7 days and 14 days after transplantation(P﹤0.01). Transplantation with SVV/GFP+BMSCs significantly inhibited inflammatory cell infiltration, scarring and collagen deposition in the infarct zone when compared with GFP+BMSCs group. Capillary density in the farct zone was 27.7% higher in SVV/GFP+BMSCs transplanted animals than in GFP+BMSCs treated animals (P﹤0.05). Further, marginal recovery of cardiac function was observed in GFP+BMSCs group. Conversely, SVV/GFP+BMSCs transplantation induced almost normal cardiac function.Conclusion SVV gene modification enhanced survival of BMSCs under ischemic condition, and transplantation with SVV engineered BMSCs may mediate substantial functional recovery after myocardial infarction.
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