| Part 1 Construction of Bicistronic Lentiviral vectorscarrying HSV-TK and EGFP geneObject To construct CMV-mediated and LEC specific promoter LEP503-mediated Lenti-HSV-tk-EGFP vector, and give a novel lentiviral vector platform for the treatment of posterior capsular opacification (PCO).Methods HSV-tk fragments from pcDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site (IRES)-EGFP to construct the bicistronic lenti-HSV-tk-EGFP transfer vector. LEP503 promoter was cloned from the genome of HLEC, and lenti-lep503-HSV-tk-EGFP was constructed by LEP503 promoter replacing CMV promoter. The lentiviral vectors were produced by transient transfection of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline (PBS) and stored at -80℃.Results Lenti-HSV-tk-EGFP can transfect several kinds of cells including primary cell and cell lines, and were stably expressed in these cells. The transfection efficiency for HLECs was about 100% at an MOI=100, and kept the samelevel for at least 6 months. The bistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector.Part 2Effects of CMV promoter -mediated Lenti-HSV-tk/GCV on Human Lens Epithelial CellsObject To investigate the cytotoxicity of lentiviral-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system on human epithelial lens cell line (HLEC) and analyze the mechanism of HSV-tk transfected cell death .Methods Using a lentiviral vector containing the Lenti-HSV-tk-EGFP as therapeutic vector and Lenti-ires-EGFP as the control. Transduction efficiency in vitro was assessed by fluorescence-activated cell sorting (FACS). Expression of HSV-tk in HLEC mediated by lentiviral vector was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed in DNA ladder and electron microscope. Time-dependent effect and bystander effect about HLEC growth inhibition induced by the HSV-TK/GCV were evaluated with CCK-8 kit.Result Transfection efficiency was more than 95%(MOI=80). When concentration of GCV is 15～25ug/ml , GCV may obviously induce infected HLEC apoptosis or necrosis. The cytotoxicity will enhance along with increasingconcentration of GCV and prolongation time. However, when concentration of GCV is higher than 25ug/ml, The growth of HLEC as a control was significantly inhibited . Nontransfected cells mixed with transfected cells were also effectively killed by GCV(bystander effect).Part 3 Selective cytotoxicity of LEP503 promoter-mediatedHSV-tk/GCV on Human Lens Epithelial Cells Object To investigate the selective cytotoxicity of lenti-LEP503-HSVtk/GCVsystem on HLEC.Methods The HLECs and Hela cell were transfected with lenti-lep503-HSV-tk, the specific expression of EGFP and HSV-tk were detected by fluorescence microscopy and RT-PCR. The HLECs were infected with Lenti-lep503-HSV-tk or Lenti-CMV-HSV-tk , the cytotoxicity of two groups were assayed and compared by CCK-8 kit.Result HSV-tk-EGFP can be specific expressed in HLEC, but the expression efficiency is less than CMV-mediated expression. Transfected cell were cultured with 20ug/ml GCV for 48 hours , and the morphologic changes of apoptosis or necrosis were seen in EGFP positive cell. The viability of Lenti-LEP503-HSVtk transfected cells were inhibited less than CMV-HSV-tk transfected cells.Part 4ATRA augment the bystander effect of HSV-tk/GCV system on Human Lens Epithelial CellsObject To investigate the enhancement of the bystand effect by the all trans— retinoic acid (ATRA) in HSV-tk/GCV system on HLEC and the correlation with expression of connexin43.Methods The cytotoxicity effect in HSV-tk/GCV in HLECs was examined by the CCK-8 kit, and the Cx43 expression was detected with Western Blot. The enhancement of bystander effect was measured by CCK-8 kits and DNA ladder.Results ATRA markedly increased sensitivity of HLECs for GCV in HSV-tk/GCV system. In the presence of 20ug/ml GCV, compared with the absence of ATRA(10-7M), an obvious decrease in survival rate was seen at any given mixture of HLECs and transfected HLECs. Western Blot showed the non-treated HLEC expressed little connexin43, but the expression of Cx43 was enhanced corresponding to the exposure time (0, 12, 24, 36, 48 hours) of ATRA(10-7M). The DNA ladder and CCK-8 results showed ATRA can significantly strengthen the bystand effect in both CMV-mediated and lep503-mediated HSV-tk/GCV system.Conclusion1 Bicistronic lentiviral vectors can be a relabe platform for gene therapy . HLEC can be infected efficiently with Lentiviral vectors coexpressing HSV-tk and EGFP.The coexpressed genes, HSV-tk and EGFP, were shown to be versatile tools for detecting the features of HSV-tk/GCV gene therapy in vitro.2 CMV-mediated Lenti-HSV-tk/GCV system can effectively kill HLECs. Thecytotoxicity will enhance along with increasing concentration of GCV and prolongation time. The bystand effect remarkably increased the cytotoxicity ofHSV-tk/GCV system.3 LEP503-mediated HSV-tk can be specifically expressed in HLECs, but the expression is lower than CMV-mediated HSV-tk/GCV. The inhibitory effect of the LEP503-mediated HSV-tk/GCV on HLECs is less than CMV-mediated HSV-tk/GCV.4 ATRA markedly increased sensitivity of HLECs for GCV in HSV-tk/GCV system. ATRA can significantly strengthen the bystander effect in both CMV-mediated and LEP503-mediated HSV-tk/GCV system. |
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