IN VIVO GENE TRANSFER ON RAT URINARY BLADDER EPITHELIAL CELL
Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants|
|Research Institution||KOBE UNIVERSITY SCHOOL OF MEDICINE|
KAMIDONO Sadao KOBE UNIVERSITY,UROLOGY,PROFESSOR, 医学部, 教授 (30030935)
HARA Isao KOBE UNIVERSITY,UROLOGY,RESEARCH ASSOCIATE, 医学部, 助手 (10263378)
ARAKAWA Soichi KOBE UNIVERSITY,UROLOGY,ASSISTANT PROFESSOR, 医学部, 助教授 (70159490)
江藤 弘 神戸大学, 医学部, 助手 (90213553)
|Project Period (FY)
1995 – 1997
Completed(Fiscal Year 1997)
|Budget Amount *help
¥4,600,000 (Direct Cost : ¥4,600,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1996 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1995 : ¥1,600,000 (Direct Cost : ¥1,600,000)
|Keywords||in vivo gene transfer / mouse urinary bladder / trans-uretheral / direct instillation / ラット膀胱 / invivo DNA導入 / 膀胱内注入|
I.IN VITRO TRANSFECTION ON PRIMARY RAT URINARY BLADDER EPITHELIAL CELL
Single 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-2
MBT-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 INSTILLATION
C3H female mouse were catheterized with 2
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 BLADDER
To 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 PRESENCE
The 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
Research Output (4results)