title: [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].
abstract: 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/Fe(3)0(4) magnetic nano-particles. 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 L02 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/Fe(3)0(4) magnetic nano-particles can significantly inhibit the proliferation of BGC823 and may become a potential biological agent for gene therapy of gastric cancer.
authors.affiliation: Department of General Surgery, Central South University, Changsha, China. wumaier@163.com
authors.forename: Wu-shouer
authors.initials: WS
authors.lastname: Buduwaili
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Bang-chang
authors.initials: BC
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D058185:Magnetite Nanoparticles; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018139:Simplexvirus / Q000235:genetics*; D013274:Stomach Neoplasms / Q000473:pathology* / Q000628:therapy; D019098:Telomerase / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
chemical_list: D058185:Magnetite Nanoparticles; D013937:Thymidine Kinase; C509186:TERT protein, human; D019098:Telomerase