[Construction of pGL3-hTERT-tk recombinant plasmid and effect of PEG-PEI/Fe(3)0(4) Nano-magnetic fluid-tk/GCV on proliferation of gastric cancer cells BGC823 in vitro].

Objective To construct an hTERT promoter-controlled recombinant plasmid HSV-tk,and to investigate its expression in human gastric cancer cells BGC823 and its effect on proliferation of gastric cancer cells BGC823 in vitro.Methods Recombinant plasmid pGL3-hTERT-tk and the corresponding reporter plasmid pGL3-hTERT-tk-Luc+ were constructed by gene engineering.The recombinant plasmids were then transfected into gastric cancer cells BGC823 via PEG-PEI/Fe3O4 magnetic nanoparticles.Fluorescence microscope was used to observe the changes of cell morphology and the transfection efficiency. The expression of the target gene in gastric cancer cells BGC823 was detected by immunohistochemistry.MTT assay was used to evaluate the effect of HSV-tk on the proliferation of BGC823 cells.Normal hepatic cells LO2 were used as controls in all the experiments.Results Recombinant plasmid pGL3-hTERT-tk was successfully constructed,and the length was 1100 bp.pGL3-control-tk-Luc+,pGL3-basic-tk-Luc+ and pGL3-hTERT-tk-Luc+ all could effectively transfect BGC823 cells with high telomerase activity,with the transfection rate being (28.1±2.3)％.Immunohistochemistry study showed significant expression of HSV-tk gene in the cytoplasm of BGC823 cells.MTT showed that 4 days after transfection of pGL3-hTERT-tk,the proliferation of BGC823 was inhibited,and the A570 value was (0.254±0.011),significantly lower than that of L02 cells (0.322-±0.013) and that of BGC823 transfected by pGL3-basic-tk (0.357±0.014) (all P＜0.05).Conclusions Magnetic nano-particles can transfect pGL3-hTERT-tk into BGC823 cells and the expression is achieved.PEG-PEI/Fe3O4 magnetic nanoparticles can significantly inhibit the proliferation of BGC823 and may become a potential biological agent for gene therapy of gastric cancer. Key words: Nano-magnetic fluids;  hTERT enhancer;  tk gene;  Gastric cancer;  BGC823 cells