Grant-in-Aid for General Scientific Research (C)
|Allocation Type||Single-year Grants|
|Research Institution||Tokyo Women's Medical College|
KIHARA Takeshi Tokyo Women's Medical College, Department of Urology, Assistant Professor, 医学部, 講師 (60195344)
TOMA Hiroshi Tokyo Women's Medical College, Department of Urology, Professor, 医学部(泌尿器科), 教授 (90075549)
ITO Fumio Tokyo Women's Medical College, Department of Urology, Clinical Fellow, 医学部(泌尿器科), 助手 (20211683)
前田 佳子 東京女子医科大学, 医学部, 助手
|Project Period (FY)
1993 – 1995
Completed(Fiscal Year 1995)
|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1994 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1993 : ¥500,000 (Direct Cost : ¥500,000)
|Keywords||DNA Transfection / thermosensitive liposome / Sendai Virus / TMGA / センダイウイルス / sendai virus|
Based on the nature of liposome as a DNA carrier and phase transfer (change in the membrane structure when heated above a specified temperature and release of substances in the liposome), experiments were performed with transfection of an organ selective gene as the final goal.
1) Preparation of STD liposome (Sendai virus fused thermosensitive liposome)
・Sendai virus was fused with LUV prepared at a lipid structure ratio of DPPC : DSPC=9 : 1, and the STD liposome was prepared.
・The phase transfer temperature was 40-41ﾟCdo which did not change after Sendai virus fusion.
・The membrane structure stability was low since there was no surface charge (about 50% of actibity lost in 1 hour).
2) In vitro study of gene transfer by STD liposome
・Human renal tubule epithelial and renal cell carcinoma lines were passaged, and transfection of PMIWZ by the liposome method was attempted.
・As a result, the expression of galactosidase was confirmed, the presence of DNA an RNA was confirmed by Southern and North
ern blotting, and transfection was confirmed.
・Expression of galactosidase was not observed under heating at 40ﾟC.
3) Gene transfer study in rat model
・A catheter was inserted into one of the renal arteries of rats, STD liposome was administered intra-arterially, and DNA expression in the injected kidney and other organs was studied with time.
・DNA expression in the injected kidney in the rat model was inadequate.
4) Neo-STD liposome preparation and in vitro gene transfer study
・Since the lack of DNA expression in rats was considered to be the cause of instability of the STD liposome membrane, TMAG (with a positive charge) was added to the structural lipid of the liposome to enhance the membrane stability and affinity to the target cells, and neo-STD liposome was prepared.
・The liposome structure of the neo-STD liposome was maintained for several hours after preparation when compared with the STD liposome.
・The same in vitro study of PMIWZ transfection as in 2) was performed using the neo-STD liposome, and the transfection efficiency was about 1%.
5) The expression of DNA in one injected rat kidney, the same as in 3), is currently being studied using the neo-STD liposome. Less