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ENHANCEMENT OF BYSTANDER EFFECTS WITH THE COMBINATION OF IMMUNO-GENE THERAPY AND PRO-DRUG GENE THERAPY

免疫基因疗法和前药基因疗法相结合增强旁观者效应

基本信息

批准号：
13671653
负责人：
KUMON Hiromi
金额：
$ 2.24万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671653/
关键词：
gene therapy pro-drug gene therapy Bladder cancer prostate cancer 5-fluorouracil Uracil phosphoribosy 1 transferas adenovirus vector Uracil phosphoribosy1 transferase

项目摘要

In this research, enhancement of bystander effects with the combination of immuno-gene therapy and pro-drug gene therapy was analyzed. As a main portion of this project, effects of UPRT (uracil phosphoribosyltransferase) gene therapy (new type of pro-drug gene therapy) for bladder cancer was analyzed. Here we show the summary of them.Objective : We evaluated whether adenovirus-mediated UPRT genes could generate sensitized antitumor effect of 5-FU on experimental bladder cancer.Material and Method : Human bladder cancer cell T24 was used for in vitro and in vivo study. Two recombinant replication-deficient adeno virus vectors, AdCA-LacZ and AdCA-UPRT, were constructed from a serotype 5 wild-type adenovirus (Ad5) by inserting CAG promoter with an E. coli LacZ gene and an E. coli UPRT gene, respectively, into the EIA region of the genome, deleting the E1B and E3 region. For in vitro chemosensitivity assay, T24 cells were seeded in 96-well plates at a density of 1000 cells/well, infected w … More ith AdCA-LacZ or AdCA-UPRT (MOI :0-100), and then exposed to 5-FU. Six days later, the sensitivity was assessed with MTT assay. For in vivo tumor treatment, subcutaneous tumors were employed by injection of T24 cells (10^7 cells). Twelve days after tumor cell injection (day 12) when tumor becomes palpable with mean estimated weight 147mg (108 -199mg), mouse were randomized to be directly injected with AdCA-LacZ or AdC A-UPRT (1X10^8 pfu/10ul) subsequently (two days later : day14) with either saline or 5-FU(2Omg/kg) intraperitoneally for 10days (from day14 to 23).Results : AdCA-UPRT infection increased the sensitivity of T24 cells to 5-FU in a MOI-dependent manner. IC50 to 5-FU shifted from 5,М in, control cells to 0.05М in AdCA-UPRT-infected cells at MOI of 50 indicating 100 fold sensitization by AdCA-UPRT infection. Consistent with in vitro studies, administration of AdCA-UPRT into the tumor of T24 cells, together with systemic 5-FU administration, resulted in sup pression of tumor growth at day28, compared with the control tumor groups : Among control groups, difference was not observed in comparis on with the presence or absence of 5-FU administrationConclusion : These results suggest that UPRT gene therapy with 5-FU can sensitize the antitumor effect of 5-FU. Consequently, this approach is a new chemosensitizing strategy for cancer gene therapy and a more feasible modality for the treatment of bladder cancer. Less

本研究分析了免疫基因治疗和前药基因治疗联合应用对旁观者效应的增强作用。作为该项目的主要部分，分析了尿嘧啶磷酸核糖转移酶（UPRT）基因治疗（新型前药基因治疗）对膀胱癌的作用。目的：探讨腺病毒介导的UPRT基因能否对5-FU产生致敏抗肿瘤作用。材料与方法：以人膀胱癌T24细胞为研究对象，进行体内外研究。以5型野生型腺病毒（Ad 5）为载体，将CAG启动子插入E. coli LacZ基因和E. coli的UPRT基因分别插入到基因组的EIA区，缺失E1 B和E3区。为了进行体外化疗敏感性测定，将T24细胞以1000个细胞/孔的密度接种在96孔板中，感染1000个细胞/孔，并在24小时内检测T24细胞对化疗药物的敏感性。 ...更多信息用AdCA-LacZ或AdCA-UPRT（MOI：0-100）处理，然后用5-FU处理。6天后用MTT法检测敏感性。对于体内肿瘤治疗，通过注射T24细胞（10^7个细胞）使用皮下肿瘤。肿瘤细胞注射后12天（第12天），肿瘤变得可触及，平均估计重量为147 mg（108 - 199 mg），小鼠随机直接注射AdCA-LacZ或AdCA-UPRT（1 × 10^8 pfu/10 ul），随后（2天后：第14天）腹腔注射生理盐水或5-FU（20 mg/kg），持续10天结果：AdCA-UPRT感染T24细胞后，T24细胞对5-FU的敏感性呈MOI依赖性增加。在MOI为50时，对5-FU的IC 50从对照细胞中的5 μ g变为AdCA-UPRT感染细胞中的0.05 μ g，表明AdCA-UPRT感染的致敏性为100倍。与体外研究一致，与对照组相比，AdCA-UPRT在T24细胞肿瘤内给药，同时全身给药5-FU，导致肿瘤生长在第28天受到抑制：在对照组中，与有或无5-FU给药相比，没有观察到差异。以上结果提示，UPRT基因治疗联合5-FU可增敏5-FU的抗肿瘤作用。因此，该方法是一种新的肿瘤基因治疗的化疗增敏策略，是一种更可行的治疗膀胱癌的方式。少