Prevent Remodeling And Repair Infarcted Myocardium By Autologous Intracoronary Mesenchymal Stem Cells Modified With AKT In Swines

Background: Transplantation of adult bone marrow-derived Mesenchymal stem cells has been proposed as a strategy for cardiac repair following myocardial damage. However cell transplantation strategies to replace lost myocardium are limited by the inability to deliver large numbers of cells that resist peritransplantation graft cell death. Accordingly, we set out to isolate and expand adult swine bone marrow-derived MSCs, and to engineer these cells to overexpress AKT1, a serine threonine kinase and powerful survival signal in many system, which can suppress apoptosis of cells, to test the hypothesis that AKT1-engineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemia swine heart.PartⅠClone of AKT1- cDNA and construct the plasmidObjective To clone the AKT gene and construct the plasmid.Methods According to the nucleotide sequence of AKT1, the CDS (regulation domin of AKT1)AKT1-cDNA fragment was amplified using RT-PCR from Hep G2 cells and the PCR product was then ligated into the pCDH1-MCS1-EF1-copGFP vector. The bacterial strain DH5αwas used as the host cell to amplify reconstructed plasmid. The amplified product was lysed to get the supernatant. Double Enzymes EcoR I and Bam H1 verified approach and analysising the sequence were used to confirm AKT-cDNA.Results AKT1- cDNA was cloned into pCDH1-MCS1-EF1-copGFP and the sequence was confirmed by comparison with the published one.Conclusion AKT1 had been successful cloned and the plasmid was reconstructed. PartⅡUsing an HIV-based lentiviral vector to transfect Mesenchymal stem cells to make them stably overexpressing AKT1Objective Using an HIV-based lentiviral vector to transfect Mesenchymal stem cells to make them Stably overexpressing AKT1 in vitro.Methods pPACKH1- Lentivector Packaging Kit with three plasmids was used to transfect Mesenchymal stem cells after constructing with pCDH1-AKT1 shuttling plasmid which amplified with transfecting 293 T cells. After 48 hours culturing, GFP(green) was detected by the fluorescence microscope. Protein was extracted for Western blot analysis and real time RT-PCR was carried out using superscript one step RT-PCR with Promega Taq kits and Sybergreen. GAPDH was used as a housekeeping gene.Results AKT mRNA expression could be detected distinctly after transfected cells 24h. Protein expression by AKT was confirmed by comparison with the published one according Western blot analysis. The expression intensity of AKT1 in MSCs remained strong 2 weeks later with detected by real time RT-PCR.Conclusion Using lentiviral vector to construct with AKT1 gene could make MSCs stably to overexpress AKT1.PartⅢSection A Culture of Mesenchymal Stem Cells from swine Bone MarrowObjective By establishing a method of the culture of swine mesenchymal stem cells(MSCs) from bone marrow, a new cell source for the cellular cardiomyoplasty is provided.Methods MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer. Results The adherent, fibroblast-like cells were confluent in single layer after plating for 10～12 days. The cell cycle analysis showed that 73% of MSCs was in G0/G1 phase.Conclusion Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. It is suggesting that MSCs may be a new cell source for the cellar cardiomyoplasty.Section B A Model of Myocardial Infarction by Intracoronary Embolization With Gelatin Sponges In SwinesObjective Producing a swine model of myocardial infarction(MI) by transcatheter embolization of the left coronary artery(LAD) with gelatin sponges.Methods Six pigs were underwent transcatheter embolization of LAD using gelatin sponge to produce anteroapical myocardial infarction. Coronary angiography,Echocardiography,MRI and Pathology were performed 4 weeks later.Results LVEDd (the LV end-diastolic dimension) increased (control versus MI: 37.0±3.4mm and 49.83±3.75 mm, p<0.01), and the ejection fraction(EF) decreased (control versus MI: 62.3±2.9% and 35.50±2.50%, p<0.001) in the MI group,. Coronary angiography revealed the LAD remained occluded. The analysis of MRI showed the same result: EDWT (the LV end-diastolic wall thick) decreased (control versus MI: 9.39±0.51mm and 6.07±1.84 mm, p<0.05), and SV(stroke Volume) decreased significantly too(control versus MI: 53.89±3.99ml and 32.81±1.55 ml, p<0.05).Conclusion This pig model of MI is reliable, repro ducible, and similar to the human condition.PartⅣInvestigate the apoptotic percentage of AKT1- MSCs and MSCs in vitro.Objective To investigate the apoptotic percentage of AKT- MSCs and MSCs and verify AKT-engineered MSCs are more resistant to apoptosis in vitro.Methods Harvesting MSCs, MSCs that only transfected with lentiviral vector (without AKT-cDNA) and AKT- MSCs, All of these cells were cultured in serum-free medium in 1% O2 at 37℃for 24 h. Then flow cytometry was used to analysis apoptotic cells after FITC- Annexin V and PI were marked them.ResultsConclusion AKT1-engineered MSCs are more resistant to apoptosis than MSCs in vitro.PartⅤthe Effect of Auto transplantation with MSCs and AKT1- MSCs for Myocardial IschemiaObjective To investigate whether implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improves cardiac function in a porcine MI model, and to explore whether the combined application of MSCs and AKT gene in the treatment of MI is feasible.Methods MI models were created by occluding the distal LAD in 24 MEISHAN pigs(21-25㎏) with gelatin sponge. The cardiac function was evaluated by magnetic resonance image(MRI) measurements and echocardiography 4 weeks later. All the pigs were divided into four groups: the control group(Group A, n=6), the DMEM group(Group B, n=6), the MSCs group(Group C, n=6), and the AKT-transfected group(Group D, n=6).With the animal under general anesthesia, 30 mL iliac bone marrow was aspirated, and the MSCs were isolated and purified by centrifuge. The MSCs were transfected with the AKT1 gene (Group D).Then autologous BrdU labeled stem cells(1×107/5 mL) was injected into LAD of the infarct heart by transcatheter in the group C and D. In the group B, 5 mL DMEM was injected in the same distraction by same approach. In group A, without injecting anything after occluding the LAD. Then after 4 weeks later, the cardiac function and regional perfusion measurements were repeated by MRI and Echocardiography , and the histological characteristics of the hearts were also studied to assess MSCs engraftment. CX-43, BrdU and VWF were tested with ELISA. VEGF, TGF-β1were analysised in the same time.Results Before the cells implantation, the LV end-diastolic dimension(EDLVd) increased and the stroke volume(SV) decreased in the MI hearts. After the cells implantation, the MRI scans showed that the cardiac function was significantly improved(compared with pre-implantation, P<0.05), and the implanted MSCs prevented the infarct region from thinning and expanding, improved contraction and increased perfusion in all groups relative to the control hearts(P<0.05). The left ventricular chamber size were smaller (P<0.05) in the hearts with being transplanted cells than that in the control hearts (P<0.05). Also, the improvement was even markedly greater in Group D (compared with Group C and B). The level of VEGF reached a high level 1 week after implanting the MSCs, but the level of TGF-β1 decreased gradually.Conclusion MSCs were capable of engraftment in host myocardium, might improve the cardiac function by attenuating contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells. Some cytokines maybe play an important role in repairing the ischemic myocardium. The overexpression AKT of MSCs might resist apoptosis that caused by the ischemia microenvironment, which was devoid of nutrients and oxygen. Then quantitation of MSCs that participating in myocardial repair was increased, and the therapeutic benefit was more powerful than that of MSCs.Combined gene transfer and cell transplantation strategies will be an effective approach for the treatment of MI.