Effects Of Lens-Specific Promoter Driven Herpes Simplex Virus-Thymidine Kinase/Ganciclovir System Enhanced By Cre/loxP System On Lens Epithelial Cells

PartⅠConstruction,packaging and purification of enhanced specific lentiviral vector combinationObjective To construct Cre/IoxP-mediated enhanced specific lentiviral vector combination(including Regulation-vector Lenti-LEP503-HSVtk-Cre and Target-vector Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk),which means lentivirus mediated specific promoter LEP503-driven Herpes Simplex Virus-Thymidine Kinase/Ganciclovir(HSV-tk/ GCV ) system of human lens epithelial cells(HLECs) and give a novel lentiviral transgenic platform.Methods Applying molecular cloning technology,Cre recombinase was cloned from Lenti-Cre by PCR and sub-cloned to T vector,which was double digested by EcoRI and SalI enzymes.At the same time,plasmid Lenti-LEP503-HSVtk-EGFP was also digested by EcoRI and SalI enzymes.The two digested sections were ligased to construct Regulation-vector Lenti-LEP503-HSVtk-Cre;Loxp-polyA-Loxp was artificially synthesized.There was PmeI enzyme-digested point at the head of polyA.When connected to T vector,it was inserted with EGFP which had PmeI enzyme-digested point at both terminal,then Loxp-EGFP-pA-Loxp vector was constructed.The Loxp-EGFP-pA-Loxp vector and plasmid PRRL were both double digested by BamHI and SalI enzymes and then directional combinated.IRES-HSVtk was cloned by PCR,which had SalI enzyme-digested point at both terminal,and it was digested by SalI enzyme together with the constructive vector.The two digested sections were ligased to construct Target- vector 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 re-suspended with 1 ml phosphate buffered saline (PBS) and stored at -80℃.HLECs were transduced by the enhanced specific lentiviral vector combination.HSV-tk expression in HLECs were evaluated by reverse transcription PCR(RT-PCR) and Western Blot.Results Most of HLECs transduced by the enhanced specific lentiviral vector combination expressed EGFR High HSV-tk expression at mRNA level was detected by RT-PCR.Western Blot showed the amount of Cre in HLECs transduced by the enhanced specific expression vector combination was the same as Cre in the Regulation-vector,EGFP was less than in the Target-vector,HSV-tk was more than in the Regulation-vector.Conclusions The enhanced specific lentiviral vector combination(including Regulation-vector Lenti-LEP503-HSVtk-Cre and Target-vector Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk) was constructed successfully and HLECs can be transduced efficiently with the vectors,which was shown to be versatile tools of HSV-tk/GCV gene therapy in vitro.PartⅡInhibitive effects of enhanced specific lentiviral vector combination/GCV system on Lens Epithelial Cells proliferationObjective To investigate whether the Cre/loxP system can enhance the inhibitive effects of lens-Specific promoter LEP503-meditated HSVtk/GCV suicide gene system on Lens Epithelial Cells proliferation.Methods The test groups were the HLECs transduced by the enhanced specific lentiviral vector combination,Regulation-vector,Target-vector or the constitutive promoter expression vector;the negative control group was the HLECs transduced by Lenti-IRES-EGFP.At the different concentration of GCV,the killing cell rates of HSVtk/GCV system on different HLEC groups were evaluated by fluorescence microscopy,flow cytometry and electron microscope.The time-dependent killing effect at different concentration of GCV was evaluated with CCK-8 kit.Results After transduced by the enhanced specific lentiviral vector combination,the inhibitory effect on HLECs enhanced along with increasing concentration of GCV and prolongation time.When concentration of GCV is 20μg/ml,GCV may obviously induce transduced HLECs apoptosis or necrosis.The inhibitive effect on HLECs in the enhanced specific lentiviral vector combination group was better than in the Regulation-vector group,but less than in the constitutive promoter expression vector group.However,when concentration of GCV is higher than 20μg/ml,the growth of HLECs as a negative control was significantly inhibited.At the concentration of 20μg/ml GCV after 96hours,the killing rate of HLECs in the constitutive promoter expression vector group detected by the flow cytometry is 93.28%, 48.58%in the Regulation-vector group,53.2%in the specific promoter expression vector group,0.96%in the negative control group,and 75.65% in the enhanced specific lentiviral vector combination group.Conclusions The enhanced specific lentiviral vector combination/GCV system can inhibit effectively the Lens Epithelial Cells proliferation.When concentration of GCV is 20μg/ml,GCV may obviously inhibit the proliferation of the infected HLECs.The cytotoxicity will enhance along with increasing concentration of GCV and prolongation time.The inhibitory effect of the enhanced specific lentiviral vector combination /GCV on HLECs is less than the constitutive promoter expression vector/GCV,but better than Regulation-vector/GCV.PartⅢSelective cytotoxicity of the enhanced specific lentiviral vector combination/GCV on Human Lens Epithelial CellsObjective To investigate the specific expression of HSV-tk and selective cytotoxicity of the enhanced specific lentiviral vector combination/GCV system on HLECs. Methods The HLECs and RPECs,NIH3T3,293T cells were transduced with the enhanced specific lentiviral vector combination.The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy and RT-PCR. The killing effects of HLECs and RPECs at the concentration of 20μg/ml GCV were assayed and compared by flow cytometry and CCK-8 kit.Results The enhanced specific lentiviral vector combination express EGFP in RPECs,NIH3T3,293T cells and most of HLECs.There was higher expression of HSVotk at mRNA level in HLEC group than in RPEC group. At the concentration of 20μg/ml GCV after 96 hours,the killing rate detected by the flow cytometry in HLEC group transduced by the enhanced specific lentiviral vector combination is 76.51%,2.24%in RPEC group. There was no significant difference in the RPE cell viability among combination-RPE group,normal-RPE group and negative-RPE control group at the concentration of 20μg/ml GCV after 72 hours(P＞0.05 ).The RPE cell viability was higher in combination-RPE group than in constitutive promoter expression vector-RPE group,which was statistically significant (P＜0.05).Conclusions The enhanced specific lentiviral vector combination express HSV-tk selectively in HLECs.It obviously inhibit the proliferation of the infected HLECs at the concentration of GCV is 20μg/ml,but has less effect on RPECs.