Enhanced Repairment Of Infarcted Myocardium By Transplantation Of Bone Mesenchymal Stem Cells Transfected With Connexin43

BackgroundAcute myocardial infarction (AMI) is a lethal and common disease.Currently there is no optimal treatment to regenerate the necrotic myocardium.Although thrombolysis and catheter-based intervention in early period after AMI can effectively open the infarct-associated vessels and save myocardium, viability of necrotic myocardium can not be restored. Recent reports have highlighted evidence on the existence of cardiac cell renewal in diseased heart,however it could not reach the demand of myocardial repairment.The infarcted myocardium can only be replaced by the fibroblasts, finally forming the noncontractile fibrous scar tissue. The loss of contractile cells may result in congestive heart failure and even death.The cellular transplantation technique gives us a good prevision to regenarate the myocardium. Lots of studies indicate that cardiac transfer of stem cells have a favorable impact on tissue perfusion and contractile performance of the infarcted heart. Several cell sources are being explored in an effort to regenerate infarcted myocardium, including fetal cardiomyocytes, embryonic stem cells, skeletal myoblasts, hematopoietic stem cells, endothelial progenitor cells, cardiac resident stem cells and mesenchymal stem cells (BMSCs). Ease of isolation, high expansion capability, inherit stability, hypoimmunogenicity and multipotent have led to the conjecture that BMSCs may be a good choice for cell-based therapies of myocardial infarction.Experimental and clinical investigations have demonstrated that there is gap junction remodeling in the marginal region around the infarcted area evidenced by abnormal distribution and down regulation of gap junction protein. Many observations have also suggested that BMSCs transplanted into the myocardium could not effectively form electromechanical coupling with host myocardiocyte.And these alterations may become the rationale of intercellular electrical uncoupling and dysrhythmiaTherefore, we put forward the hypothesis that BMSCs modified with connexin43,a gap junction protein,may better integrated with the host myocardiocyte and finally enhance repairment of the infarcted myocaium.Objective1. To optimize the method for isolation, purification and cultivation of rat BMSCs.2. To explore the possibility and superiority of Lentivirus as a vector in animal transgenesis.3. To observe the therapeutic effect of the homogeneously exogenous transplantation of rat BMSCs on treatment of myocardial infarction.4. To determine whether myocardial repairment is enhanced by BMSCs modified with connexin43 gene.Part I Isolation,cultivation and identification of rat BMSCsMethod We examined the isolation, purification, expansion in vitro.Bone marrow mononuclear cells were isolated with adherence screening method. BMSCs were obtained by removing the non-adherent cells, and cultured in low glucose DMEM with 10% fetal bovine serum. Then the BMSCs were purified and expanded through passaging in time. The cells were identified with flow cytometry, light microscope.Result The rat BMSCs were expanded for five passages, 1x108 cells were obtained. The FACS results showed that BMSCs expressed antigen CD90 and CD44,the symbol of stem cell,and did not express CD34,CD45 and CDl4. The large quantity, the high amplification potential and the effective method of purification play key roles in exclusive differentiation of BMSCs. Conclusion The conditions of isolating, cultivating, purifying and proliferating human BMSCs were optimized in present study,enough BMSCs was obtained for furthermore research.Part II Expression of Human Connexin43 Gene in Bone Mesenchymal Stem Cells Mediated by Lentiviral VectorMethod The connexin43 gene was recombined to a HIV-1 lentiviral vector with EGFP by infusion technique.The combined plasmid was identified by sequencing.The three-plasmid system of lentiviral vector was consisted of FUGW, the packaging plasmid, and the envelope plasmid VSVG, which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles. The rBMSCs were infected by obtained lentiviral particles. The expression of connexin43 protein and infection rate was observed with fluorescent microscope. We adopted hemiquantitative RT-PCR to analysis the expression of connexin43 mRNA. Result The result of sequencing showed that the cloned connexin43 gene was consistent with the sequence reported in GeneBank. The insertion of connexin43 gene in viral genome was confirmed by PCR. The FUGW-Connexin43 plasmid was identified the correct sequence.After the three plasmids of lentiviral vectors were cotransfected to the 293T cells,the supernatant was collected and concentrated ,the titer of the lentiviral vector particles was found to be 1x 108TU / mL. Connexin43 gene-modified rBMSCs show linear and stippled patterns of Cx43 expression on cell membrane. RT-PCR showed that the expression of Cx43mRNA in gene modified BMSCs were higher than that of the control group.Conclusion Lentiviral vector carrying connexin43 gene has been successfully constructed.The infected rBMSCs are able to express the connexin43 protein. Connexin43 gene-modified rBMSCs show linear and stippled patterns of Cx43 expression on cell membrane.This will facilitate the following exploratory development of connexin43 gene modified stem cell-based cardiac repairment. Part III Bone Mesenchymal Stem Cells Transfected with Connexin43 Transplantation for Myocardial Infarction in RatsMethod AMI was produced in male SD rats by ligating the left coronary artery. The 30 male rats were randomly divided into three groups:â‘ Group A,the animals transplanted with connexin43-EGFP gene modified BMSCs,n=10;â‘¡GroupB, the animals transplanted with EGFP gene modified BMSCs,n=1;0â‘¢GroupC, the animals injected with saline,n=10; The rats received exogenous transplantation of BMSCs 0.5-1 hour after myocardial infarction (1X 106 BMSCs/100u1, injected at four points); The animals were sacrificed 4 weeks after coronary artery ligation and the heart function and other indices were evaluated. Result Compared with GroupC,the systolic function of groupA and groupB was significantly improved, respectively(P<0.01,each), the difference between groupA and groupB was significant(P<0.01).Infarct size of groupA and groupB was diminished Compared with the GroupC (P<0.01), especially the groupA. RT-PCR and western-blot showed that Cx43 mRNA and the protein depressed in groupB and GroupC,however the Cx43 mRNA and the protein were significantly increased in groupA. The transplanted cells distribute widely in ischemic and infarcted regions and their nucleus show the oval shape as those of myocardiocyte, the arrangements of the donated cells were paralleled with host myocardium fibers in host hearts and the connexin43-EGFP gene modified BMSCs was closed to host myocardiocyte,which may indicate the formation of gap junction. Conclusion Allogeneil graft BMSCs to repair infracted myocardium was feasible and efficacious. Connexin43 gene modified BMSCs could further improve the cardiac function after myocardial infarction compared with the EGFP/BMSCs transplantation.Connexin43 gene modified BMSCs may form gap junction with host myocardiocyte.Summary1. Lentiviral vector carrying connexin43 gene has been successfully constructed.The rBMSCs could be genetically modified with connexin43 gene by a lentiviral vector.2. We demonstrate for the first time that expression of connexin43 in BMSCs was diminished after transplanted into the infarcted myocardium,the down-regulation could be reversed with the connexin43 gene modification.3. Connexin43 gene modified BMSCs could further improve the cardiac function after transplanted into the infarcted myocardium,it may due to the overexpression of connexin43.