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Y. Lu, J. Yang; Effects of Bicistronic Lentiviral Vectors Mediated HSV–tk/GCV System on Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2539.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the cytotoxicity of lentivirus–mediatedHSV–ck/GCV suicide gene therapy system on human epitheliallens cell line (SRA 01/04)and study the mechanism of cell death.
HLEC was infected with lentiviral vectorsco–expressingHSV–tk and EGFP or expressing EGFP alone and then treatedwith GCV. The transduction efficiency in vitro was assessedby FACS. Expression of HSV–tk in HLEC mediated by lentiviruswas examined by fluorescence microscope, genomic PCR and RT–PCR.The cytotoxicity of HSV–tk/GCV suicide–gene systemwas assessed in DNA ladder and electron microscope. Time effectand bystander effect of HLEC growth inhibition were evaluatedwith Cell counting kit–8 .
The lentivirus canmediate stable integration and efficient expression of HSV–tkin HLEC with transduction efficiency higher than 95%(Fig 1).At concentration of 15∼25ug/ml , GCV significantly induces apoptosisor necrosis in HLEC infected by Lenti–HSVtk–EGFP.The cytotoxicity is enhanced along with increasing concentrationof GCV and prolongation time.(Fig 2) There was no obvious toxicityon the cells infected by Lenti–EGFP,used as a control(P<0.05). Uninfected cells mixed with lenti–HSVtk–EGFP–infectedcells were also effectively killed by GCV .
GCVcan effectively kill HLEC expressing HSV–tk.The bystandereffect remarkably increased the cytotoxicity of HSV–tk/GCV.Bicistronic lentiviral vectors can efficiently integrate severalgenes into HLEC and may serve as a reliable platform for genetherapy. Therefore,lentivirus mediated HSV–tk/ GCV suicidegene therapy system may provide an effective approach to thetreatment of posterior capsule opacification.
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