Expression Of Gene Connexin43 Enconded Into Eukaryotic Expression Vector In Skeletal Myoblast Cells

ObjectiveOne common character in acute myocardial infarction and heart failure is loss of function cardiomyocytes due to necrosis and apoptosis. Currently to lack a guts myocardial to hurt of cure main concentration in improvement myocardial of the blood provide, easing the symptom that the myocardial lacks blood, but these can't make myocardial cell rebirth and avoid the occurrence of heart failure. So cell replacement therapy to replace cardiomyocytes irreversibly lost may prove a promising strategy to regenerate myocardium. Currently , these are many kinds of seed cells,including fetal cardiomyocytes,myoblasts, embryonic and adult stem cell. Skeletal myoblast cells may be considered best candidate for its easy availablility,no requirement of immunosuppression,easily transfected and no requirement of ethics. For implanted cells to actually contribute to the synchronous contraction of the heart ,however,they must be anatomically integrated with the exisiting host myocardium. Connexin43 is the major gap junction protein in the heart responsible for intercellular communication between cardiomyocytes.So the integrity of connexin43 in stucture and function plays an important role in coupling between grafed cells and host cells. Although gap junctions exist between undifferetiated myoblast with current expression of connexin43,they are absent from mature skeletal muscle with little connexin43 expression.In the study,we transfected the skeletal myoblast cells with the eukaryotic expression vector which encodes full length connexin43 cDNA.Then,we investigate the transfected cells with methods of immunocytochemical staining, western-blot and laser scanning confocal microscope.Methods1 pure the eukaryotic expression vector which encodes full length connexin43 cDNAThe eukaryotic expression vectors encoding full length connexin43 cDNA are amplified in E.coli DH-5α.Then the plasmids were pured with knit of tiangen PureFection Plasmid DNA Purification System and knit of Wizard? PureFection Plasmid DNA Purification System.2 the L6 rat skeletal muscle cell line cultureThe L6 rat skeletal muscle cell line was grown in Dulbecco's modified eagle's medium supplemented with 15% fetal calf serum.Skeletal myoblasts can be incubated without differentitaition if cultured in this medium at 60%--70% fuse.3 density of G418When the density of G418 is higher than 400mg/ml,all the cells would die in the presence of G418 in 14 days 4 gene transfer and selectionThe transfection of connexin43 was mediated by Lipofectamine2000. After 24 hours the cells were plated at a limiting dilution (1cell/ well)to obtain single-cell clononies in the presence of G418(250mg/ml).After 14 days the surviving cells, which were stably transfected and thus show long-term protein expression ,were picked up and grown in number . 5 immunocytochemical staining and western blotting to assess connexin43 protein expression5.1 immunocytochemical staining of connexin43 protein expressionThe cells which were stably show protein expression were investigated with immunocytochemical staining .Then the optium desity of positive cells was caculated with image analyzed software.5.2 western blotting to assess connexin43 protein expression The connexin43 protein expression was analyzed with Western blotting . connexin43- or control-transfected cells were grown as protocol described next.Results1 the discrimination of the eukaryotic expression vector encoding connexin43 cDNAElectrophoresis result indicated the former brand was about 1.7kb,matching connexin43 gene ;the latter brand was about 6.9 kb matching the vector.And the plasmids had high purity and density. 2 L6 rat skeletal muscle cell line cultureL6 cells look liked shuttles. L6 cells fused into skeletal myotube when they got in touch with each other. 3 density of G418When the density of G418 is higher than 400mg/ml,all the cells would die in the presence of G418 in 14 days. 4 the result of gene transfer and selectionSingle-cell clononies were obtained.Then these single-cell clononies grew in number.5 immunocytochemical stainning for connexin43 and western blotting to assess connexin43 protein expression5.1 immunocytochemical stainning for connexin43Transfected cells demonstrated high-level connexin43 expression. Control transfected cells demonstrated low-level connexin43 expression. The ash degree of transfected cells was higher than control-transfected cells which was caculated with image analyzed software.5.2 western blotting to assess connexin43 protein expressionThe lines were subjected to westen blotting for connexin43,demonstrating that all of them expessed higher level of connexin43 than control L6 cells.The level of connexin43 which the obtainedcell lines expressed were 2.138±0.098 fold and 2.236±0.168 fold, P<0.05Conclusions1 We had generrated connexin43-overexpression skeletal myoblast cell lines that resulted in improved formation of functional intercellular gap junctions. Then the cell lines was used to investigate the role in communication between gafted cells and host cardiomyocytes.2 The method of transfection for skeletal myoblast cells was impoved according to the characters of this research. Then the cell lines stably showed connexin43 protein expression were obtained.3 According to the results of this researsh,we can get higher transfectin efficiency with lipofectamine2000 than Calcium phosphate.