Anti-proliferative Effect Of Pamam-d Deliveried Double Suivide Gene Transfer System On Human Tenon's Capsule Fibroblasts In Vitro

Objective:Double suicide gene pAcGFP1-Hyg-TK-CD mediated by nanoparticals PAMAM dendrimers which is non-immunogenicity as non-virus delivery to transfect Human Tenon's capsule fibroblasts (HTFs), and observe it's killing effect to HTFs and 'bystander effects', in order to look for a safe and effective method for HTFs inhibition modulates after glaucoma filtration surgery.Methods:1. Construct the expression plasmid vector of pAcGFP 1-Hyg-TK-CD with PCR,enzyme restriction and ligation. The vector was ideniified by enzyme cutting and Sequencing.2. EGFP reporter gene was transfected into HTFs using the second-and fifth-generation PAMAM-D and Lipofectamine 2000 respectively.The surface properties of vectors were evaluated with transmission electronic microscope, dynamic laser scattering and zeta potential analyzer. Detect the protection effect to DNA from DNase digestion. A number of variables for optimal transfer of the transfection complexes were tested, including concentrations of plasmid DNA, weight ratios of DNA:vector and dendrimer generations.3. The HTFs were cultured in vitro,and identified by means of morphological, histological and immunocytochemical methods. Nanoparticals PAMAM-D mediated plasmid pAcGFP1-Hyg-TK-CD with TK and CD was used to deliver HTFs, it was detect the expression of TK and CD in HTFs by means of RT-PCR and Westen-blot respectively.4. Detect cytotoxicity and lethal effec of GCV and 5-FC to HTFs cells by MTT assay. Select the optimal concentration of GCV and 5-FC which are low cytotoxicity and high killing effect to HTFs-TK-CD. To observe the morphology changes of transfected HTFs cells after use GCV and 5-FC by light microscope and transmission electronic microscope. Detect the apoptosis rates of HTFs by flow cytometry,and also detect the "by stander effect'of double suicide gene.Results: 1. We got 1617bp fragments through enzyme cutting.The result of sequencing was equal to the expection.2. Our reserch demonstrated that relatively high buffer capacities of PAMAM-D occurred at PH ranges is from 5 to 10. The average size of DNA/dendrimer complexes was below 100nm, and showed an excellent resistance against DNase I digestion. Variables such as concentrations of plasmid DNA and dendrimer generations influenced the transfection efficiency to some extent, whereas the weight ratios of complexes exhibited the most significant effects on transfection level. Optimal weight ratios of G2-PAMAM,G5-PAMAM,Lipid that DNA/dendrimer complexes are 8:1,2:1,4:1, The transfection efficiency of G5 PAMAM-D was higher than of G2 AMAM-D (42.1% vs 19.4%,P<0.05) at optimal weight ratios.3. The HTFs were cultured successfully in vitro. It is identified by RT-PCR and Western-blot that the mRNA and the protein expression of double suicide gene TK and CD respectively.4. The concentration of GCV 3μg/ml,5-FC 200μg/ml is the optimal choise of prodrugs.The light microscope showed that the growth of untransfected HTFs were in good condition, there were no obvious changes of cell population and cell proliferation, the changes with the combination of GCV and 5-FC were no difference from single prodrugs; however, the growth condition of HTFs which transfected double suicide gene went bad and the cells number decreased, especially treated with both GCV and 5-FC.Transmission electronic microscope shows cell apoptosis phenomenon in HTFs.After use GCV 3μg/ml,5-FC 200μg/ml and GCV 3μg/ml+5-FC 200μg/ml in HTFs-TK-CD group, the apoptosis rate is 11.86%,22.37% and 33.13% respectively. Prodrug of GCV and 5-FC not only could kill the transfected HTFs, but also has'by stander effect'.Conclusion:1. Gene engineering technique were used to construct the expression plasmid vector of pAcGFP1-Hyg-TK-CD with CD and TK. The vector was ideniified by enzyme cutting and Sequencing.2. Nanoparticals PAMAM dendrimers is safe and low cytotoxicity, with relatively large PH ranges and high buffer capacities, it was also shown an excellent protection to DNA. The weight ratios of complexes play a key role in transfection efficiency, variables such as concentrations of plasmid DNA and dendrimer generations influenced the transfection efficiency to some extent.3. Double suicide gene TK and CD mediated by nanoparticals PAMAM-D was transfected HTFs, it was detect the expression of double suicide gene TK and CD in HTFs by means of RT-PCR and Westen-blot respectively4. Prodrugs GCV and 5-FC were used in suitable concentration after TK and CD were transfected the HTFs, The concentration of GCV 3μg/ml,5-FC 200μg/ml is the optimal choise of prodrugs.The prodrug could be trnsfered into cytotoxicity material to kill HTFs in vitro, the killing effect that combination of GCV and 5-FC were stronger than single one, it also has'by stander effect'. We can believe that this study will provide a sound basis for glaucoma filtertion surgery.