Mutation Of Hypoxia Inducible Factor 1α May Improve Graft Autologous Mesenchymal Stem Cells Survival In Ischemic Heart Disease

Background: In recent years, with the emergence of stem cell engineering, more and more attention was paid to mesenchymal stem cells in areas of the treatment of ischemic heart disease. Research shows that stem cells can differentiate into cardiac cells, a function of increasing the number of cardiac cells, increasing the density of blood vessels regional myocardial infarction, improving heart function. While the survival of stem cells depends on the cultivation of the micro-environment, the low stem cells survived rate of myocardial ischemia transplant is one of the important factors on the clinical efficacy. It was confirmed that HIF--1α(hypoxia-inducible factor 1α, HIF-1α) generated by hypoxia can inhibit apoptosis. However HIF-1αdegrades very fast, and can not meet the needs of myocardial ischemia and hypoxia. Therefore, we use two-way fixed-point mutation in the reorganization of HIF-1α, then transfer stem cells in vitro and in vivo studies in the hypoxic conditions. We investigated whether the gene mutation in HIF-1αstem cells can inhibit apoptosis, improve treatment efficacy. Furthermore the mechanism of autologous stem cell transplantation of gene transfer treatment in ischemic cardiomyopathy was also investigated.PartⅠSection A The isolation, and purification of swine autogenous bone marrow-derived mesenchymal stem cellsObjective : To evaluate the culture and purification of bone marrow-derived mesenchymal stem cells in vitro.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 10～14 days. The cell cycle analysis showed that 75% 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 SwinesObject: Producing a swine model of myocardial infarction(MI) by transcatheter embolization of the left coronary artery(LAD) with gelatin sponges.Method: Six swines were underwent transcatheter embolization of LAD using gelatin sponge to produce anteroapical myocardial infarction. Coronary angiography,and Pathological examination were performed 4 weeks later.Result: In myocardial infarction (MI) group, left ventricular developed pressure (LVDP) decreased compared with the control group (P<0.05),while±dp/dtmax also declined(P <0.05). LAD coronary angiography review prompted embolization still exists. In MI group, transmural infarction was formed in anterior and septal.Conclusion The model is stable, reliable and much closer to clinical pathology physiological processes. Use of gelatin sponge can successfully establish coronary occlusion swine myocardial infarction model. It has the advantages of minor trauma, animal survival for a long time and easily feeding after surgery.PartⅡConstruction of pcDNA3.1+-GCA-HIF-1α-GCCObject: Construct HIF--1α(hypoxia inducible factor-1, HIF-1α) eukaryotic expression vector pcDNA3.1 +-GCA-HIF-1α-GCC, the mutant of pcDNA3.1 +-HIF-1α, and pack it with Lentivirus.Method: PcDNA3.1 +-HIF1αof HIF1αNo. 567 Proline HIF1αPro codon was mutated into the CCA alanine (Ala) codon GCA with site-directed mutagenesis approach, building into a single mutation HIF1αeukaryotic expression vector pcDNA3.1 + HIF1α– 567Ala in the first mutation. On the basis of that,site-directed mutagenesis was used to mutated pcDNA3.1 + - HIF1α-567 Ala No. 811 in amide (Asn) codon for AAC alanine (Ala) codon GCC,building into dual site-directed mutagenesis HIF1αeukaryotic expression vector pcDNA3.1 +-GCA-HIF-1α-GCC.RT-PCR and Western blot were used to detect the expression of HIF1αin the transferred cells. Recomposed Lentivirus vector of HIF1αwas digested and sequenced, then packed into the reorganization HIF1αLentivirus virus in 293 cells. PCR was used to identify HIF1αexpression. Titer of virus vector was determined.Results: Human double-mutant HIF-1αeukaryotic expression vector GCA-HIF-1α-GCC was successfully constructed by identification of digestion and gene sequencing. Reorganization of Lentivirus was packaged successfully, which was tested by PCR. The virus titer is 2 .5×108tu/ml.Conclusion: Human double-mutant HIF-1αeukaryotic expression vector GCA-HIF-1α-GCC was successfully constructed to express the corresponding protein and mRNA, which lay the foundation for gene therapy of HIF-1αfor ischemic heart disease.PartⅢLentivirus mediated GCA-HIF-1α-GCC gene transfer of bone marrow stem cells and the detection of apoptosisObject: Observe the GCA-HIF-1α-GCC expression and it affection to the growth of cells in the Lentivirus mediated GCA-HIF-1α-GCC gene transfer of swine MSCs. Detection of apoptosis rates respectly in the GCA-HIF-1α-GCC gene transferred bone marrow stem cells, blank transferred bone marrow stem cells and primary cells in the hypoxic environment.Method: After GCA-HIF-1α-GCC-cDNA shuttle plasmid transfected pCDH1 packaging cell 293T cells were amplified, gene was recombined by the four plasmid congested slow virus gene transfer system and transferred into swine bone marrow stem cells. It's expression was detected by fluorescence ion. Western blot was used to detect protein expression. After FITC-Annexin V and PI double-stained, cell apoptosis and death of MSCs, blank transferred MSCs and GCA-HIF-1α-GCC transferred MSCs in 1% O2, 37℃in the incubator for 24 h were detected by flow cytometry analysis.Western blot was used to identificae protein expression of Bax and HO-1.Results: 24 or 48 hours after transferring gene into 293 T cells and MSCs, strong fluorescence can be detected. Western blot showed that the molecular weight of aimed protein is consistent with the known molecular weight of HIF-1α. RT-PCR real-time quantitative results showed that two weeks after the transfer, MSCs can stable transcript HIF-1α. Primary cells in hypoxia and lack of nutritional status undergoing a lot of apoptosis, while the apoptosis rate is 61.3 percent compared to the GCA-HIF-1α-GCC transferred MSC of 7.1 percent. A large number of deaths occurred in the blank transferred cells, while the mortality rate is 67.3 percent. Western blot was used to identificae protein expression of Bax and HO-1.Conclusion: Transfer of portable GCA-HIF-1α-GCC gene with the Lentivirus transfer of viruses into bone marrow stem cells of swine can stable express HIF-1α. The GCA-HIF-1α-GCC transferred MSCs in vitro showed more tolerance to apoptosis induced by lack of nutrition hypoxia than simple MSCs. It is because maybe that the stable expression of HIF-1αenhance the secretion of HO-1 of stem cells, which suppress Bax expression.PartⅣThe experimental study of GCA-HIF-1α-GCC-modified autologous bone marrow stem cell transplantation in the treatment of ischemic cardiomyopathyObject: To investigate whether GCA-HIF-1α-GCC-modified autologous bone marrow stem cell transplantation could significantly improve cardiac function in myocardial ischemic heart disease with swine MI model. Further, to discuss the MSCs and gene combined therapy in myocardial ischemia and the possible mechanism involved.Method: 24 small Meishan swines from 15 to 20 kg , divided into four groups: A group: blank group, B group: DMEM group, C group: MSCs Group and Group D: TG group. Myocardial infarction LAD Model is established by gelatin sponge embolization. Four weeks later, hemodynamics and MRI were used to assess heart function. 30 mL bone marrow was taken from the iliac of each swine, separated and purificated by methods of density centrifugal and adherent screening. MSCs were amplified in vitro, while MSCs of D Group transfected with GCA-HIF-1α-GCC gene. Nano-iron marked autologous bone marrow stem cells (1×107 / 5 mL) injected transcatheter into the remote embolization of LAD. After one week, two weeks, four weeks, six weeks and eight weeks of injections of MSCs, Powerlab and MRI were used to assess cardiac function and stem cell marker. Plasma BNP was detected by ELISA in before infarction and stem cell transplantation or after that. We estimated risk zone of myocardial infarction and use Western blot to test PPAR. Results: Before transplantation, MRI showed the left ventricular end-diastolic diameter (EDLVd) increased, stroke volume (SV) decreased significantly, hemodynamics LVDP and±dp / dtmax dropped significantly in cardiac infarction.Cardiac function of C, D group has improved after transplantation (P<0.05). The transplanted MSCs prevented thinning and expansion of infarct zone. Compared with Group A and B , the contract function and perfusion had improved (P <0.05). This improvement in cardiac function in Group D is more obvious (vs the C group, P<0.05). Group D increased more significantly than group C. BNP of C, D group has decrease after after transplantation (P<0.05), Group D increased more significantly than group C(P<0.05). The level of VEGF reached a high level 1 week after implanting the MSCs,then decreased gradually.Conclusion: MSCs can survive in the host myocardium and improve left ventricular function by preventing thinning of infarction zone and inhibit contraction dysfunction. Overexpression of GCA-HIF-1α-GCC can make transplanted stem cells more tolerant of microenvironment hypoxia and lack of nutrient in the early transplantion, reduce apoptosis and increase absolute number of stem cells participation in cardiac rehabilitation, which can improve heart function more significantly. Application of GCA-HIF-1α-GCC genes combined with MSCs autologous transplantation is an effective way to the treatment of ischemic cardiomyopathy.