[The effect of lentivirus-mediated suicide gene therapy on lens epithelial cells].
OBJECTIVE
To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death.


METHODS
a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated.


RESULTS
the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect).


CONCLUSIONS
GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.
Yang
Jin
J
Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.
Lu
Yi
Y
Guo
Li-he
LH
Liu
Tian-jin
TJ
Mo
Xiao-fen
XF
D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D015774:Ganciclovir / Q000494:pharmacology*; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016086:Lentivirus; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection
D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir