【作者】 秦卫军；

【导师】 陈宝琦；

【作者基本信息】 第四军医大学 ， 泌尿外科， 2001， 硕士

【摘要】 膀胱癌是泌尿系中最常见的肿瘤，在我国发病率居泌尿系肿瘤首位，并且有恶性度高，易复发的特点。目前治疗以手术、化疗为主，但复发、转移率仍较高，晚期疗效差，需积极探讨新的治疗方法。 自杀基因疗法是目前有效治疗肿瘤中最有前景的基因治疗策略之一。葡萄糖醛酸苷酶（β-Glucuronidase，βG）是溶酶体中的酸性水解酶，参与机体的生理、病理及药物代谢等过程。它能够特异性水解葡萄糖醛酸苷糖甙键，释放出葡萄糖醛酸和配基。βG在ADEPT（抗体导向酶/前体药疗法）的应用研究中，已证实其前体药对肿瘤的选择性杀伤作用。将βG及前体药系统与基因治疗相结合，就有可能产生新的能够定向实施分子化疗的方法，用βG的相应前体药作用于βG高表达膀胱肿瘤，有可能起到杀伤肿瘤作用。为膀胱癌的治疗寻找一条新的、有效的途径。本实验进行了以下几方面研究： 1、利用质粒PGEM-4及pcDNA3.1（+）具有相同的EcoRI酶切位点，成功构建带βG基因的真核表达载体PcDNA3.1（+）-βG。 2、利用脂质体（LipofectAMINE）介导法将含有全长βG cDNA的真核表达载体pcDNA3.1（+）-βG转染至人膀胱癌细胞T24中，经G418筛选，获得了稳 第四军医大学硕士学位论文定转染 6 G基因的细胞克隆；通过 RNA打点杂交及原位杂交和免疫荧光、免疫组化、wes七ern Blot方法分别从帕A及蛋白水平证实p G基因己经稳定整合转入T24细胞基因组中并获得p G高表达，将稳定转染并高表达p G的转染细胞系命名为：T24什 G细胞。并通过光镜、电镜、细胞增殖周期检测等方法观察了p G转染的 T24／fi G细胞的生物学特性。 3、对pG前体药Epi－bUS作用于T24／pG细胞的体外效应进行了一系列研究，结果显示：稳定转染6 G基因的 T24／e G细胞对 Epi十US的敏感性比亲本细胞提高了 1000倍以上，IC。／半数抑制剂量）为 0．179／L。浓度为 2．929／L的Epi－b［JS作用4－5天对转染细胞的生长影响很小，但可将高表达p G的转染细胞全部杀死；0．2925／L的ESi－bus也能在6－7天内将转染细胞全部杀死，表现出明显的“自杀效应”；将T24／日G细胞与其末转染的亲本细胞T24以不同比例混合，观察了 Epi－blls对混合细胞的作用，结果示：当 T24旭 G细胞占川％～20％的时候，有70％～90％的混合细胞被杀死，说明有较强的旁观者效应。 上述研究表明：pG基因叩G前体药物系统可引入自杀基因治疗系统，脂质体－真核表达载体介导日G基因转染，结合使用日G前体药物，有望成为治疗膀脱癌的有效策略，为该系统治疗膀耽癌的临床应用提供了理论和实验依据。更多还原

【Abstract】 Bladder carcinoma is the most common malignancy in the urinary, accounting for 20% of the malignancy of all the systems. The managements remain to be surgery and chemotherapy. High frequent recurrence and bad efficiency in the late stage call for new therapies for it. The suicide gene therapy is one of the most promising gene therapies for the tumor. f3-Glucuronidase is a kind of acid hydrolase in lysosome, involved in the processes of physiology, pathyology and metabolism of the drugs. It can hydrolysis glucuronide glycoside bond, releasing the glucuronic acid and ligand. It was well established that the prodrug of 13G can selectively kill the tumor cells. By combining the j3G, prodrugs and the gene therapy, a new oriented molecular chemotherapy may be developed. The bladder tumor cells with high expression of ~G take the prodrug and will be killed selectively. The aim of this study is to look for a new and effective therapy for the bladder carcinoma. The experiment was done in the following ways: 1 .The retrovirus eukaryotic expression vector with the f3G gene was constructed by the same EcoR I site of the PGEM-4 and pcDNA3.1(+). 2.By the lipofectAMlNE, a retrovirus eukaryotic expression vector pcDNA3.1 (+) containing the whole length sequence of the f3G cDNA was transfected into the human bladder tumour cell line T24. After the G41 8 screening, a clone that get the stable transfection of the BG was obtained. It was confirmed that the ~G gene had been stably integrated into the genomic DNA of the 124 cell and got a highly expression by the means of RNA blotting, in situ hybridization, Immunoflurescence, Jmmunohistochemistry, Western Blot. The stably transfected cell was named T24/ f3G. The biological characteristics of the fG transfected cell were studied by the methods of microscopy and electron microscop. 3.The series studies of the in vitro effects of the prodrug Epi-bus on T24/ f3G showed that the stably transfected cells are more sensitive to the Epi-bus by 1000 fold than the parent, with the IC50 0.1 7g/L. The Epi-bus with a concentration of 2.92g/l sustaining for 4-5 days had little affection on the growth of the transfected cells, but will kill all the cells with high j3G expression. The concentration of 0.292g11 for 6-7 days will kill the transfected cells, showing the obvious uicide effects The T24/G cells were mixed with parental cells not transfected with various proportion and 70%-90% of the mixed cells were killed when the 124/ f3G cell accounting for 1 0%-20%. It showed the potent stand-by effect. The study showed that the 13G gene and its prodrug could be introduced into the suicide gene system and the j3G gene transfection mediated by the LipofectAMlNE- retrovirus eukariotic expression vector combining the 13G prodrug managment will be the promising strategy for bladder therapy.更多还原