IN VIVO GENE TRANSFER ON RAT URINARY BLADDER EPITHELIAL CELL

点击翻译按钮获取中文摘要

I.IN VITRO TRANSFECTION ON PRIMARY RAT URINARY BLADDER EPITHELIAL CELLSingle cell suspensions of Fischer-344 rat urinary bladder primary epithelial cells were obtained. Cells were cultured to appropriate confluency for transfection. Transfection efficiency was measured by BETA-galactosidase staining assay 48 hours after pSVBETAgal plasmid DNA transfection with a) calcium phosphate precipitation and b) lipofection.a) Calcium phosphate precipitation : 1 xl0^3 cells of lx10^7 had BETA-gal expression for mean.b) Lipofection : 5x10^3 cells of 1x10^7 for mean.II.IN VITRO TRANSFECTION ON MBT-2MBT-2, mouse bladder cancer cell, were cultured to appropriate confluency and were tranfected pSVbgal plasmid DNA as mentioned above.a) Calcium phosphate precipitation : 1x10^4 cells of 1x10^7 had b-gal expression for mean.b) Lipofection : 4x10^4 cells of 1x10^7 for mean.III.IN VIVO GENE TRANSFER TO MOUSE BLADDER EPITHELIUM WITH DIRECT TRANS-URETHERAL INSTILLATIONC3H female mouse were catheterized with 2 … More 2G intravenous needle trans-uretherally, instillated with serum containing pSVBETAgal plasmid DNA-Iiposome complex with 2 hour clump. Mouse bladder were resected 48 hour after the removal of urethral clump and frozen sections were assayed with X-gal solution over night. Transfection efficiency was not so sufficient that only 2 or 3 positive cells were observed per each section. The difference between the positive rate in vitro and in vivo assay demonstrated the importance of pre-treatment on mouse bladder.IV.P RE-TREATMENT ON MOUSE BLADDERTo establish successful gene transfer, we performed several trans-uretheral pre-treatment on mouse bladder. a) DMSO : DMSO/PBS treatment of several concentration was performed trans-uretherally 2 hour before DNA challenge. Transfection efficiency was not high as we expected. b) PEG6000, c) adriamycin were also challenged. c) Adriamycin demonstrated most high efficiency to show 7 to 15 positive cells per each section. This indicates that inflammation will present higher trnasfection efficiency in vivo, still in vivo gene transfer is not sufficient for clinical trial.V.CONCLUSION AT PRESENCEThe difference of transfection efficiency between in vitro and in vivo assay indicated that gene therapy upon bladder cancer or another urothelial tumor shold not be taking part of conventional therapy at this time. Some other pre-treatment or trial of adenoviral infection are undergoing. Less