Construction And Expression Of Eukaryotic Expression Vector PcDNA3.0-Cx43 In Mesenchymal Stem Cells

One common character in acute myocardial infarction and heart failure is loss of functioning cardiomyocytes due to necrosis and apoptosis.It is widely accepted that cardiomyocytes are terminally differentiated cells that exhibit very low capacity to regenerate.So cell replacement therapy to replace cardiomyocytes irreversibly lost may prove a promising strategy to regenerate myocardium.Historically,there are many kinds of seed cell,including fetal cardiomyocytes,myoblasts,embryonic and adult stem cells.Of these, autologous marrow stromal stem cells may be considered best candidate for its multipotentiality,easy availability,and no requirement of immuno-suppression.For implanted cells to actually contribute to the synchronous contractions of the heart,however,they must be anatomically integrated with the existing native myocardium.Gap junction is mainly responsible for intercellular communication between cardiomyocytes.Connexin 43 is the major gap junction protein in the ventricular myocardium reponsible for intercellular communication between cardiomyocytes and is therefore likelyto have an important role in couling between grafted cells and hostcardiomyocytes.In this study,we construct an eukaryotic expression vector encodingfull-length connexin43 cDNA, transfect marrow stromal stem cells with thevector and investigate Cx43 overexpression in mesenchymal stem cells.Methods 1) Full-length connexin43 cDNA was amplified in E.coli JM109and cloned into the eukaryotic expression vector pcDNA3.0. The vectorpcDNA3.0-Cx43 was then constructed.2) rat mesenchymal stem cells wereseparated and cultured in vitro.3) Stably transfected cell colonies wereobtained by gene transfer and G418 screening-Expression of cx43 gene wasverified with immunocytochemistry and Western Blotting.Results Electrophoresis results indicated the recombinant vector wasconstructed correctly.Immunocytochemical analysis revealed anoverexpression of connexin43 in pcDNA3.0-Cx43 transfected MSCs screenedby G418 in comparison with control- transfected MSCs.Conclusions 1) Percoll separating medium is a well-referenced medium fordensity gradient centrifugation of MSCs;2) Eukaryotic expression vectorpcDNA3.0-Cx43 was constructed expectively,3) Protein connexin 43 can beexpressed in mesenchymal stem cells...