[Inhibitory effects of HSV-tk/GCV system mediated by recombinant adeno-associated virus 2 on rabbit lens epithelial cells].

OBJECTIVE: To study the inhibitory effects of recombinant adeno-associated virus 2 (rAAV2)-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on the rabbit lens epithelial cells (N/N1003A) in vitro and to investigate the mechanism of cell death. METHODS: After N/N1003A cells had been transfected with rAAV2-EGFP, expression of enhanced green fluorescent protein (EGFP) were observed by inverted fluorescent microscope and the transfection efficiency was detected by flow cytometry. N/N1003A cells were infected by recombinant virus rAAV2/HSV-tk as the treated group, and the uninfected N/N1003A cells were used as the controls. The dose- and time-dependent efficiency and bystander effect of HSV-tk/GCV system on the cells were studied by MTT assay. Apoptosis and necrosis were observed by phase contrast microscope, electron microscope and Hoechst33258 stain. Apoptotic cell rate and cell cycle were detected by flow cytometry. RESULTS: rAAV2 vector encoding EGFP gene could be transfected into N/N1003A cells stably and efficiently. The effects of GCV on these two groups were dose-dependent (F = 13.076. 239, P < 0.001). The difference of percentages of survival cells between the study group and the control group at various doses of GCV was statistically significant (F = 53,47.119, P < 0.001). The 50% of the inhibitory concentration (IC50) of GCV in the study group was 2 mg/L and was 524 mg/L in the control group. The killing efficiency of GCV increased with the prolongation of time and showed significant bystander effect. Cell apoptosis and necrosis were observed in N/N1003A-tk cells transfected by GCV, and the percentage of apoptotic cells was significantly higher than that of the control group (t = 3.83, P < 0.01). The percentages of N/N1003A-tk cells in the S phase of the cell cycle was significantly higher than that of the control group (t = 3.55, P < 0.01). Whereas the percentages of the G0/ G1 phase in GCV treated cells was significantly lower than that of the control group ( t = 4.29, P < 0.01). CONCLUSIONS: GCV can kill efficiently the N/N1003A cells infected by recombinant virus rAAV2/HSV-tk, and there is strong bystander effect. Recombinant adeno-associated virus-mediated HSV-tk/GCV suicide gene system may provide an effective approach for the treatment of lens posterior capsular opacification.

[Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene].

OBJECTIVE: To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification. METHODS: The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR. RESULTS: The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells. CONCLUSION: The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.