title: [The cell-type-specific HSV-tk gene expression of suicide gene therapy system regulated by lens-specific promoter LEP503].
abstract: OBJECTIVE
  To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEP503.
  
  
  METHODS
  Experimental research. The enhanced specific HSV-tk/GCV gene system of two vectors were constructed (Lenti-LEP503-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk). The lentiviral vectors were produced by transient transduction of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80°C. The HLEC and RPEC, NIH3T3, 293T cells were transduced with the enhanced specific HSV-tk gene system. The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy, flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow cytometry and CCK-8 kit. Difference of RPE cell viability among groups was evaluated by analysis of variance (ANOVA).
  
  
  RESULTS
  Expression efficiency of EGFP in RPEC group was 62.3%, 68.3% in NIH3T3 group, 75.8% in 293T group, whereas 17.5% in HLEC group. There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group. The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01, whereas 0.29 in RPEC group. At the concentration of 20 mg/L GCV after 72 hours, the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSV-tk gene system was 76.51%, and 2.44% in RPEC group. There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group, normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours (MD(1) = -0.047, P = 0.671; MD(2) = 0.027, P = 0.912).
  
  
  CONCLUSIONS
  The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC. At the concentration of 20 mg/L GCV, it is effective against the proliferation of HLEC in vitro, but has less kill effect on RPEC.
authors.affiliation: Department of Ophthalmology, Fudan University, Shanghai, China.
authors.forename: Yong-xiang
authors.initials: YX
authors.lastname: Jiang
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.forename: Tian-jin
authors.initials: TJ
authors.lastname: Liu
authors.forename: Jin
authors.initials: J
authors.lastname: Yang
authors.forename: Xin-hua
authors.initials: XH
authors.lastname: Wu
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics*; D004847:Epithelial Cells; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D051379:Mice; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D055213:Retinal Pigment Epithelium / Q000166:cytology; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C431569:LENEP protein, human; D013937:Thymidine Kinase