Objective To investigate a change in the differentiation and biological function of the cultured rat fibroblast (FB) transfected by the myoblast determining gene (MyoD) and the connexin 43 (Cx43) gene and to explore the possible mechanism of the MyoD and Cx43 genes on treatment of ischemic heart disease (IHD). Methods The gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and pLenti6/V5DEST-Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. The RFL-6 FB cells were transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, followed by the Blasticidin (50 μg/ml) selection, according to the lentiviral expression system (ViraPower) protocol. The expression and the biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, and molecular and immunocytochemical methods. The mophological structure changes of the cells were observed under microscope before and after the transfection. Results The expression of MyoD and Cx43 was detected in the MyoD and Cx43 genes transfected FB with RT-PCR and Western blot. The immunocytochemical methods indicated the expressionsof the MyoD and Cx43 genes, while desmin and αactin were found in these cells. The myotubes were found from the cultures incubated a week in the differentiation medium, in which the transfected cells had a characteristic of the filamentsin their cytoplasm and showed a myoblast morphology. Conclusion MyoD cDNA can induce the cultured FB to differentiate into the myoblasts and Cx43 cDNA can enhance the gap junctional intercellular communication between the cell and the cell. Thus, a further experimental foundation for the therapy of IHD can be provided.
Objective To observe the influence of connexin 43 (Cx43) on bystander effects induced by cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) coexpression suicide genes system in human cholangiocarcinoma QBC939 cells and transplantation tumors in nude mice. Methods In vitro, the CD+tk+ and CD+tk+Cx+ cells were respectively treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV). The cytotoxic efficacy was evaluated by microculture tetrajolium test (MTT) method. In order to investigate the influence of Cx43 on bystander effects, the volumes of transplantation tumors of the CD+tk+ and CD+tk+Cx+ cells were measured before and after application of 5-FC and GCV. Results CD and tk gene were stably expressed in transfected QBC939 cells. Increasing expression of Cx43 was determined by testing for the presence of Cx43 mRNA by RT-PCR and the presence of Cx43 protein by Western blot in CD+tk+Cx+ cells. The killing effect of 5-FC and GCV on CD+tk+Cx+ cells was more effective than that on CD+tk+ cells both in vitro and in vivo. Conclusion Double suicide genes system CD/5-FC+tk/GCV could induce remarkable killing effect on cholangiocarcinoma cells in vitro and transplantation tumors in vivo. The cotransfection of Cx43 gene is able to enhance the bystander effects and the inhibition of carcinoma cells.