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IN VIVO GENE TRANSFER ON RAT URINARY BLADDER EPITHELIAL CELL

大鼠膀胱上皮细胞体内基因转移

基本信息

批准号：
07457373
负责人：
KAMIDONO Sadao
金额：
$ 2.94万
依托单位：
KOBE UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457373/
关键词：
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

项目摘要

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

I.对原代大鼠膀胱上皮细胞的体外转染获得Fischer-344大鼠膀胱原代上皮细胞的单细胞悬浮液。将细胞培养至适当的汇合度用于转染。在用a）磷酸钙沉淀和B）脂质体转染pSVBETAgal质粒DNA后48小时，通过BETA-半乳糖苷酶染色测定来测量转染效率。a）磷酸钙沉淀：1 × 10 ^7中的1 × 10 ^3个细胞平均具有BETA-gal表达。B）脂质体转染：将MBT-2（小鼠膀胱癌细胞）培养至适当的汇合，并如上所述转染pSVbgal质粒DNA。a）磷酸钙沉淀：B）脂质转染：平均值为1 × 107个细胞中的4 × 104个细胞。III.用直接经尿道灌注法向小鼠膀胱上皮进行体内基因转移 ...更多信息 2G静脉注射针经尿道滴注含pSVBETAgal质粒DNA-脂质体复合物的血清，2小时成团。在去除尿道块后48小时切除小鼠膀胱，用X-gal溶液过夜测定冷冻切片。转染效率不够，每个切片仅观察到2或3个阳性细胞。体外和体内试验中阳性率之间的差异证明了对小鼠膀胱进行预处理的重要性。IV.对小鼠膀胱的预处理为了建立成功的基因转移，我们对小鼠膀胱进行了几次经尿道预处理。a）在DNA攻击前2小时，经尿道进行几种浓度的DMSO：DMSO/PBS处理。转染效率不如预期。B）PEG 6000，c）阿霉素也被攻击。c）阿霉素表现出最高的效率，每个切片显示7至15个阳性细胞。这表明，在炎症反应中，体内基因转染效率较高，但体内基因转染尚不足以进行临床试验。5.结论目前，体内和体外转染效率的差异表明，目前对膀胱癌或其他尿路上皮肿瘤的基因治疗不应作为常规治疗的一部分。其他一些腺病毒感染的预治疗或试验正在进行中。少