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Development of suicide bomb bectors and its effective transfer for cancer therapy.

自杀式炸弹袭击者的发展及其对癌症治疗的有效转移。

基本信息

批准号：
17591352
负责人：
YAMADA Osamu
金额：
$ 2.24万
依托单位：
Tokyo Women's Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591352/
关键词：
cancer cell suicide bomb telomerase gene transfection

项目摘要

The promoter of telomerase is active in virtually all types of tumors but is silent in most adult somatic cells. We placed the suicide genes under the control of the telomerase promoter with the aim of restricting their expression to tumor cells. As the suicide genes, we used 1. the herpes simplex virus thymidine kinase gene which may confer ganciclovir sensitivity to all tumor cells and 2. bovine α1-3 galactosyltransferase (α1-3GT) cDNA which produces the αGal epitope. The carbohydrate epitope, αGal epitope, is known as a major xenoantigen and the epitope exists abundantly in non-primate mammals. Human and Old world monkeys have anti-αGal antibody, as a natural antibody. The transfection of the functional α1-3GT gene driven by telomerase promoter into human cancer cells may lead to their transformation, making them susceptible to lysis by natural antibodies.(1) We made the construct of the herpes simplex virus thymidine kinase gene under the control of the telomerase promoter with the … More aim of restrinting its expression to tumor cells. In transfecton experiments, the telomerase promoter driven tymidine kinase gene (hTERTp-TK) conferred ganciclovir sensitivity to leukemic cell line tested, whereas nomal somatic cells remained largely unaffected. Human hTERTp-TK positive K562 cells implanted in nude mice developed into tumors that could be eradicated by ganciclovir treatment.(2) The fragment encompassing the whole coding sequence of bovine α1-3GT cDNA including the tanslation start site was subcloned to PEGFP basic vector which had been inserted with telomerase promoter. Bovine α1-3GT cDNA under the control of telomerase promoter (hTERTp-α1-3GT) was electrophoretically transfected into the human leukemic cell lines. Stable transfomant was obtained by selection with G418 and, limiting dilution technique. The expression of the αGal epitope was confirmed by flow cytometry, using specific binding with IB4 lectin conjugated with fluorescein isothiocyanate. I have been examining whether the leukemic cells expressing the αGal epitope can be lysed by natural antibodies and this is true for another cancer cell lines. Less

端粒酶的启动子在几乎所有类型的肿瘤中都有活性，但在大多数成人体细胞中是沉默的。我们将自杀基因置于端粒酶启动子的控制下，目的是限制它们在肿瘤细胞中的表达。作为自杀基因，我们使用1。单纯疱疹病毒胸苷激酶基因，其可赋予更昔洛韦对所有肿瘤细胞的敏感性;和2.牛α1-3半乳糖基转移酶（α1-3GT）cDNA，其产生αGal表位。糖表位αGal表位是已知的主要异种抗原，并且该表位在非灵长类哺乳动物中大量存在。人和旧大陆猴都有抗αGal抗体，作为一种天然抗体。将端粒酶启动子驱动的功能性α1-3GT基因转染人癌细胞可能导致其转化，使其对天然抗体的裂解敏感。(1)我们构建了在端粒酶启动子控制下的单纯疱疹病毒胸苷激酶基因， ...更多信息目的是抑制其在肿瘤细胞中的表达。在转染实验中，端粒酶启动子驱动的胸苷激酶基因（hTERTp-TK）使白血病细胞对更昔洛韦敏感，而正常体细胞基本上不受影响。人hTERTp-TK阳性K562细胞移植于裸鼠体内后可形成肿瘤，经更昔洛韦治疗后可被根除。(2)将牛α1-3GT cDNA全长编码区（含翻译起始位点）亚克隆到已插入端粒酶启动子的PEGFP基础载体上。将端粒酶启动子调控下的牛α1-3GT cDNA（hTERTp-α1-3GT）转染人白血病细胞系。经G418筛选和有限稀释技术，获得了稳定的产酶菌株。通过流式细胞术，使用与异硫氰酸荧光素缀合的IB 4凝集素的特异性结合来确认αGal表位的表达。我一直在研究表达αGal表位的白血病细胞是否能被天然抗体裂解，这对另一种癌细胞系也是如此。少