In Vitro Transfection Of Rat Bone Marrow Mesenchymal Stem Cells With Recombinant Adenovirus Vector Containing Human HCN4Gene: An Experimental Study

ObjectiveTo construct recombinant adenovirus vector Ad-hHCN4-IRES/eGFP andtransfect the vector to rat bone marrow mesenchymal stem cells (BMSCs), andto detect the expression of hHCN4mRNA and protein of the target gene intransfected BMSCs. In order to confirm hHCN4gene can expression in BMSCs,and to progress confirm it can provides the basis for the BMSCs who carrieshHCN4gene and has the pacing function，and laing the foundation for building acombination of gene and cell biological pacing therapy.Methods1. The bone marrow mesenchymal stem cells were separated and purifiedand cultured passage by density gradient centrifugation and adherent culturemethod；The surface antigen were identified by immune cells fluorescencemethod.2. The recombinant adenovirus vector Ad-hHCN4-IRES/eGFP (the purposeof the genome) carring hHCN4gene was builded successfully and wastransfected into rat bone marrow mesenchymal stem cells in vitro.3. After the transfection, the expression of green fluorescent protein wasobserved in BMSCs by using fluorescence microscopy.4. After the transfection, the expression of mRNA was detected in BMSCs byRT-PCR method. 5. After the transfection, the expression of hHCN4gene protein wasdetected in BMSCs by Western Blot method.Results1. BMSCs were isolated、purified and adherent passaged successfully.Under an inverted microscope, we seed the BMSCs afrer the primary culturedadherent and most of them was the clone-like growth of fibroblast-like spindlecells and growth in good condition after passage；Immune cells fluorescencesee the expression of surface antigen CD90、CD44of BMSCs were positive, therate were96.74%and94.36%respectively.2. The purpose of the genome was successfully transfected into rat BMSCs；BMSCs which expressed hHCN4gene were obtained.3. Under a fluorescence microscope, after24h transfection we observed theexpression of green fluorescent protein in BMSCs and there was no significantdifference in the expression and distribution of green fluorescent protein;transfection efficiency was about10-20%.4. After the transfection, BMSCs which carried the target genome wasdetected by RT-PCR method and in BMSCs hHCN4mRNA was expressed.5. After the transfection, BMSCs which carried the target genome wasdetected by Western Blot method and in BMSCs the level of hHCN4geneprotein was expressed.Conclusions1. BMSCs was successfully isolated and purified and passaged by densitygradient centrifugation and affixed to the wall culture method.2. After the transfection, BMSCs which carried the target genome wasdetected Using fluorescence microscopy and by RT-PCR、Western blot method.The recombinant adenovirus vector Ad-hHCN4-IRES/eGFP carried hHCN4gene in vitro can be successfully transfected into rat BMSCs and the BMSCscan express hHCN4mRNA and protein.