Transplantation Of Connexin43-overexpressing Bone Marrow Mesenchymal Stem Cells In Post-infarction Heart Failure Rat

Objective:To construct, package and purify the recombinant lentivirus carrying green fluorescent protein (GFP) gene and the rat connexin 43 gene (Cx43).Methods:The rat Cx43 gene was amplified by RT-PCR and connected to the lentiviral expression vector pGC-FU, which is carrying the EGFP gene, to construct the lentiviral vector plasmid pGC-FU-Cx43. Restriction digestion analysis and DNA sequencing was used to verified the correction. The viral particles were generated by cotransfection of 293T cells with the pGC-FU-Cx43 and two packaging vector (pHelper1.0, pHelper2.0) and the virus titer was determined by counting the percentage of GFP positive cells.Result:The insert orientation of the Cx43 gene in the lentiviral vector plasmid pGC-FU-Cx43 was verified by restriction digestion analysis.Target Cx43 sequences was confirmed by the bulk sequencing. The final titer obtained was 2x109TU/ml.Conclusion:The pGC-FU-Cx43 recombinant lentivirus carrying GFP gene and Cx43 gene with high viral titer was constructed and packaged successfully and would pave the way for the further study in vitro and in vivo. Objective:Transfecting pGC-FU-Cx43 recombinant lentiviral into amplified rat BMSC to observe the Cx43 expression level and the function change of intercellular communication in BMSC.Methods:Rat BMSCs were isolated and purified by density gradient centrifugation and adherent cell culture. Flow cytometry was used to identify the surface markers in P3 cells. After transfection of pGC-FU-Cx43 recombinant lentiviral into P3 cells, the expression of EGFP was observed by fluorescence microscope, the level of Cx43 protein in BMSC was determined by western blot assay and the changes of cell communication was measured by FRAP assay.Results:High purity BMSC was obtained after isolation and purification. Increased EGFP expression was observed, expression level of Cx43 protein was significantly increased, and communication between cells was remarkably enhanced in pGC-FU-Cx43 transfected BMSC.Conclusion:The combination of density gradient centrifugation and adherent cell culture method can isolate BMSC efficiently. pGC-FU-Cx43 recombinant lentiviral was transfected into BMSC successfully to increase the expression of target protein and enhance the communication between cells. Objective:The goal of the present study was to explore the effect of transplantation of bone marrow mesenchymal stem cells over-expressing Cx43 on heart failure in post-infarction rats.Methods:120 rats were randomly divided into four groups:sham group consisting of 30 rats, DMEM/F12 group consisting of 30 rats injected with DMEM/F12, EGFP group consisting of 30 rats transplanted EGFP transfected BMSC, and Cx43 group consisting of 30 rats transplanted Cx43 transfected BMSC. Myocardial infarction models were built by ligating the anterior descending branch and then the cells were transplanted after 30 minutes. The rats were sacrificed and heart function was measured by echocardiography after 4 weeks. Cardiac electrophysiology examination was operated in the Langendorff isolated heart perfusion system and then tachycardia arrhythmia was induced.Results:Compared with DMEM/F12 group, heart function was improved significantly, myocardial infarct size reduced and collagen fiber content was decreased significantly in EGFP group and in Cx43 group especially. After myocardial infarction, expression of Cx43 in myocardial tissue reduced significantly, but increased after transplantation of BMSC over-expressing Cx43. Survival BMSC and the formation of gap junction between BMSC and the host myocardium could be found using confocal laser both in EGFP group and Cx43 group. Electrophysiological examination showed that arrhythmia prone to be induced in DMEM/F12 group. There was no significant difference in EGFP group and DMEM/F12 group. The rate of arrhythmia induced in Cx43 group was lower than that in DMEM/F12 group and EGFP group.Conclusion:Transplantation of allogeneic BMSC attenuated ventricular remodeling, improved cardiac function, and had no significant impact on induced arrhythmia. Furthermore, heart function after myocardial infarction could be improved significantly. and the rate of arrhythmia induced could be reduced significantly in Cx43 group transplanted BMSC which over-expressed Cx43. This effect may result from the overexpression of Cx43 gene which can improve gap junction remodeling, and increase electro-mechanical coupling between BMSC and the host myocardial cells.